Detection of Functionally Active Melanocortin Receptors and Evidence for an Immunoregulatory Activity of -Melanocyte-Stimulating Hormone in
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《内分泌学杂志》
Department of Dermatology and Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin (M.B., M.E., Z.L., S.W.S., V.O., T.A.L.) and Department of Medicine, Hematology, and Oncology (S.D.), University of Münster, D-48149 Münster, Germany
Department of Pediatrics and Genetics and the Howard Hughes Medical Institute (G.S.B.), Stanford University School of Medicine, Stanford, California 94305
Department of Dermatology (A.V., K.S., U.B.-P.), Center for Applied Cutaneous Physiology, University Hospital Charité, D-10117 Berlin, Germany
Abstract
Proopiomelanocortin (POMC)-derived peptides and their receptors have been identified in many peripheral organs including the skin in which they exert a diversity of biological actions. We investigated the expression and potential role of the POMC system in human dermal papilla cells (DPCs), a specialized cutaneous mesenchymal cell type regulating hair follicle activity. In culture, these cells expressed POMC and displayed immunoreactivity for ACTH, MSH, and -endorphin. Among the prohormone convertases (PCs) tested, only PC2, its chaperone 7B2, and furin convertase but not PC1 and paired basic amino acid cleaving enzyme 4 gene were detected. Human DPCs in vitro expressed both the melanocortin-1 receptor (MC-1R) and MC-4R, and immunoreactivity for these receptors was also present in cells of the human dermal papilla in situ. In contrast to the dermal papilla of agouti mice, agouti signaling protein, a natural and highly selective MC-1R and MC-4R antagonist, was undetectable in human DPCs. The MC-Rs detected in human DPCs were functionally active because MSH increased intracellular cAMP and calcium. Preincubation of the cells with a synthetic peptide corresponding to the C-terminal domain of agouti signaling protein abrogated cAMP induction by MSH. Furthermore, MSH was capable of antagonizing the expression of intercellular adhesion molecule-1 induced by the proinflammatory cytokine interferon-. Our data suggest a regulatory function of MSH within the dermal papilla whose disruption may lead to deregulation of immune and inflammatory responses of the hair follicle, thereby possibly contributing to the development of inflammatory forms of alopecia.
Introduction
THE PROOPIOMELANOCORTIN (POMC) (1) system is an evolutionary conserved regulatory module that is expressed in the brain but also in many peripheral tissues including the skin (1, 2). In the skin the POMC system is crucially involved in a diversity of physiologic processes such as pigmentation, inflammation, lipogenesis, and exocrine gland activity. Because the skin expresses all regulatory components of the POMC system and responds to stressful stimuli with induction of this system, it has been suggested that an equivalent of the hypothalamic-pituitary-adrenal axis is operational in the skin (1).
POMC contains within its structure the amino acid sequences of several hormonal products including the melanocortins, which are ACTH, MSH, MSH, and MSH as well as the endogenous opioids -, - and -endorphin (ED). Generation of these hormones from POMC requires the action of at least two precursor or prohormone convertases (PCs) belonging to the family of serine proteases. Processing of POMC at dibasic residues is mediated by either PC1 alone (resulting in ACTH and -lipotropin) or a combination of PC1 and PC2 plus the PC2-specific binding protein 7B2 yielding MSH and ED (3). There is recent evidence from in vitro and in vivo data that additional PCs such as paired basic amino acid cleaving enzyme 4 gene (PACE4) or furin convertase may also process POMC (3, 4). Whereas ED elicits its biological effects via binding to opioid receptors, the melanocortins mediate their biological actions by binding to melanocortin receptors (MC-Rs). Five MC-Rs (MC-1R to MC-5R) have been cloned belonging to the superfamily of G protein-coupled receptors with seven transmembrane domains (5). They differ in their relative binding affinity to ACTH and the various MSH peptides (5, 6).
With regard to the hair follicle, it is known for many years that MSH affects the coat color of mice (7). Subsequent studies provided accumulating evidence that many components of the POMC system including the upstream regulators, CRH and its receptor, are expressed in the pilosebaceous unit of mouse skin (8, 9, 10, 11, 12, 13). In these studies POMC and POMC-derived peptides exhibited differential spatial and temporal expression in the pilosebaceous unit and the hair follicle, i.e. expression varied, depending on the hair cycle. In mice, the functional significance of melanocortins in coat color regulation is highlighted by the fact that animals with a signaling-deficient MC-1R (recessive yellow mutation) display yellow pigment due to the shift of eumelanin toward pheomelanin synthesis (14). On the other hand, mice ubiquitously expressing agouti protein (AP), a natural antagonist of MC-1R and MC-4R, have a completely yellow fur (15). It was also reported that in vivo administration of certain melanocortins to animals can modulate hair growth. In the mink, anagen hair growth, i.e. the phase of the hair cycle during which synthesis of hair takes place, can be induced by intracutaneous injection of ACTH but not by MSH (16). When injected into murine telogen skin (the quiescent phase of the hair cycle), ACTH has a similar anagen-inducing effect (17). Immunohistochemical studies on the back skin of neonatal C57BL/6 mice and adolescent C57BL/6 mice with depilation-induced hair cycle further showed that MSH and MC-1R immunoreactivity are differentially expressed in various cells of the hair follicle during hair follicle morphogenesis and cycling, suggesting a role of melanocortins beyond hair follicle pigmentation (18).
In man, genetic studies have corroborated the functional role of the MC-1R for hair color. It was shown that northern European populations with red hair and sun sensitivity display common MC-1R alleles including the Arg151Cys, Arg160Trp, and Asp294His (19). Regarding the ligands for MC-1R, it was further demonstrated that POMC mutations leading to trace amounts of POMC-derived peptides or complete absence of ACTH, MSH, and ED result in red hair (20). Finally, it is also known that therapeutic administration of ACTH can lead to hypertrichosis and hirsutism (21).
Although these findings suggest that the human hair follicle is a target site for melanocortins, expression and function of the POMC system in this adnexal structure of the skin remained incompletely characterized until recently. In this report we investigated whether human dermal papilla cells (DPCs), a unique myofibroblast cell type involved in hair follicle activity, express components of the POMC system. Our studies revealed that various elements of the POMC system are expressed in DPCs established from human skin. The expressed MC-Rs are functionally active because prototypical responses such as cAMP and calcium signaling can be elicited by MSH. Finally, our studies on the immunoregulatory action of MSH in human DPCs emphasize a functional aspect that may be of particular relevance in the pathogenesis of inflammatory forms of alopecia and/or its future therapy.
Materials and Methods
Establishment of human DPC cultures
Isolation and culture of human DPCs was performed as reported previously (22). The Institutional Review Board of the University Hospital Charité, Berlin, had previously approved all of the described studies. All experiments adhered to the Declaration of Helsinki guidelines. DPC cultures were established from normal scalp tissue of healthy patients undergoing plastic surgery for face lifts or from the margins of unaffected scalp skin after surgical excision of benign cutaneous lesions. Scalp skin was collected in DMEM with 20% fetal calf serum (FCS), 100 U/ml penicillin/streptomycin, 0.4 mM [SCAP];l-glutamine, and 50 ng/ml amphotericin B (all from Biochrom, Berlin, Germany). Complete anagen follicles were microdissected under a stereomicroscope (47 50 20; Carl Zeiss, Jena, Germany) according to the technique of Jahoda and Oliver (23) as modified by Reynolds and Jahoda (24). Each dermal papilla was cut across the stalk region, transferred to a 35-mm culture dish, and incubated in a humidified atmosphere with 5% CO2 at 37 C. Confluent DPC cultures were subcultured after 8 wk and propagated in the culture medium as described above. DPC cultures were used at passage numbers 2–4. Cell cultures were usually discontinued after five passages.
RNA extraction, RT-PCR, and sequencing
Total RNA was prepared using the RNeasy kit (QIAGEN, Santa Clarita, CA). After DNA digestion with DNAse (Promega, Madison, WI), 2 μg of total RNA were reverse transcribed with 15 U AMV-RT (Promega) and amplified with primers against MC-1R to MC-5R, PC1, PC2, 7B2, and POMC under identical conditions as reported (25, 26, 27). PACE4 was amplified with primer sets established by others (28). All amplification products were of the expected size. For the detection of the 111-bp amplification product of furin convertase, the following primers were used: forward, 5'-TGC TGG TCT TCG TCA CTG TC-3' and reverse, 5'-TTG TAG GAG ATG AGG CCA CGG-3'. The amplification protocol was 1 x 94 C for 5 min; 60 C for 1 min; 72 C for 2 min; 35 x 94 C for 45 sec; 60 C for 2 min; 72 C for 1 min; 1 x 94 C for 45 sec; 60 C for 1 min; and 72 C for 10 min. To confirm the identity of the latter product, the respective band was extracted from the 2% agarose gel using an extraction kit from QIAGEN. The extracted DNA was cloned into a pGEM-T easy vector (Promega) and sequenced (4base lab GmbH, Reutlingen, Germany). Expression of agouti signaling protein (ASIP) was performed as reported previously with slight modifications (29). The primers were: forward, 5'-ATGGATGTCACCCGCTTACT-5' and reverse, 5'-GCGGAAGAAGCGGCACTGG-3', generating a 385-bp amplicon. The amplification protocol was 1 x 94 C for 5 min; 60 C for 1 min; 58 C for 2 min, 72 C for 2 min; 31 x 94 C for 45 sec; 58 C for 1 min; 72 C for 2 min; 1 x 94 C for 45 sec; 58 C for 2 min; and 72 C for 10 min. As a positive control for ASIP, microdissected normal human testis derived from routine surgical procedures (kindly provided by Dr. C. Poremba, Department of Pathology, University of Düsseldorf, Düsseldorf, Germany) was used.
Quantitative real-time PCR
For RNA expression studies of intercellular adhesion molecule (ICAM)-1, DPCs were deprived for 24 h in 1% FCS followed by stimulation with 100 ng/ml interferon (IFN)- alone or in combination with 10–6 M -MSH (Bachem, Bubendorf, Switzerland) for 4 h. In some experiments cells were also stimulated with 10–8 M MSH. Quantification of mRNA levels was carried out by real-time fluorescence detection as previously reported (25, 30). Primers and probes for ICAM-1 and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from PE Applied Biosystems (Foster City, CA) (GAPDH: Hs99999905; ICAM-1: HS00164932-M1). cDNA was amplified by PCR in the ABI Prism 7700 sequence detector (PE Applied Biosystems). 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, Darmstadt, Germany) 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 normal human dermal fibroblasts. The relative amounts of gene expression were calculated by using GAPDH as a standard. Each PCR analysis was performed in triplicates from a total of three independent experiments. Data were expressed as means ± SD and analyzed by the Student’s t test.
Determination of cAMP
DPC (2 x 104 cells/well) were seeded into 96-well tissue culture plates in regular culture medium. On the next day, cells were thoroughly washed and switched to RPMI 1640 without FCS. Cells were subsequently deprived from FCS for 48 h followed by stimulation with MSH for 20 min in the presence of 0.1 mM isobutyl methylxanthine (IBMX). In some experiments IBMX was also preincubated for 1 h before stimulation with MSH. In another set of experiments, DPCs were stimulated with MSH for 10 and 40 min. For competitive blocking experiments, a synthetic peptide containing the amino acids 87–132 of the C-terminal domain of ASIP (Phoenix Pharmaceuticals, Belmont, CA) was incubated with the cells at 10–6 M 15 min before stimulation with MSH at the same concentration. In all experiments forskolin at 0.1–1 μM served as a positive control. Cells were subsequently lysed and cAMP was determined by a specific enzyme immunoassay (Amersham Pharmacia Biotech, Freiburg, Germany). Triplicate wells were used for each treatment and statistical analysis was performed using the Student’s t test.
Calcium measurements
For detection of intracellular calcium levels, DPCs were trypsinized, plated on glass coverslips, and grown in regular culture medium for 1–2 d. Subconfluent cell monolayers were then deprived from FCS for 48 h. Cells were incubated with 4.5 μmol/liter of the fluorescent dye FURA2-AM in HEPES-buffered Ringer solution (HBRS) containing (in millimoles per liter): 140 NaCl, 5 KCl, 1 MgCl, 1 CaCl2, 5 glucose, 10 HEPES adjusted to pH 7.4 for 15 min at 37 C. Then they were washed three times with FURA2-free HBRS. Coverslips with loaded cells were placed in a temperature-controlled (37 C) perfusion chamber and mounted on the stage of inverted microscope (Diaphot 300; Nikon, Tokyo, Japan). About six cells were focused. Excitation wavelength alternated between 340 and 380 nm, and emitted fluorescence was monitored at 535 nm by a microscope photometer (hardware and software for controlling and achieving alternating excitation and data acquisition obtained by Photon Technology International, Hamburg, Germany). The tracings were corrected in each experiment for autofluorescence before calculating the 340:380 ratio. During the experiment, cells were continuously superfused with HBRS. For calcium measurements the perfusion media were supplemented with MSH at various concentrations ranging from 10–6 to 10–10 M. As positive control, inonomycin at 10–4 M was used.
Western immunoblotting
Total cell lysates were prepared by scraping the cells into lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM ethylenglycol-bis(-aminoethylether)-N,N,N'N'-tetraacetic acid and 0.01% NaN2 plus freshly added proteinase inhibitors (10 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). After sonication and centrifugation, supernatants were analyzed for protein content using the modified Bradford assay (Bio-Rad Laboratories, Richmond, CA). An equal volume of 2x sodium dodecyl sulfate loading buffer was added and the samples were boiled at 95 C for 5 min. Equal protein amounts were separated by denaturing SDS-PAGE using 4–12% gradient gels from NuPAGE (Invitrogen, Carlsbad, CA) followed by immunoblotting. The polyvinylidene difluoride membranes were blocked overnight with 10% BSA. For the detection of immunoreactive POMC, an anti-ACTH antibody (Sigma, St. Louis, MO) directed against the amino acids 18–39 of ACTH (1:1000). For the detection of ASIP, sample preparation and SDS-PAGE were carried out under nonreducing conditions. Generation of the rabbit polyclonal anti-AP antibody directed against the full-length (His23-Cys131) of AP is described elsewhere (31). The used anti-AP antibody detects 1 ng/lane of the immunogen and cross-reacts with ASIP (31). Protein lysate from human testis derived from routine surgical procedures (kindly provided by Dr. C. Poremba, Department of Pathology, University of Düsseldorf, Düsseldorf, Germany) served as a positive control for ASIP. Membranes were incubated with primary antibodies overnight, washed, and incubated for 20 min with a horseradish-peroxidase-conjugated secondary antibody (1:10,000; Amersham Life Science, Freiburg, Germany). Antigen-antibody complexes were visualized with the enhanced chemiluminescence kit (Amersham). To confirm identical loading of lysate proteins the original blot was stripped as reported before (32) followed by reblocking and incubation with an anti--tubulin antibody (1:1000; Oncogene Research Products, San Diego, CA).
Immunofluorescence studies
DPCs were seeded into 8-well Lab-Tek Permanox chamber slides (Nalge Nunc International, Naperville, IL) at a density of 10,000 cells/well. On the following day, cells were either fixed with 4% paraformaldehyde for 20 min at room temperature (for the detection of plasma membrane MC-1R and MC-4R immunoreactivity) or subsequently permeated with 0.3% Triton X-100 for 10 min (for the detection of cytoplasmic MC-1R and MC-4R immunoreactivity). Nonspecific binding was blocked with 5% donkey serum for 1 h. Cells were then incubated for 1 h with either a rabbit anti-MC-1R antibody (at 1–5 μg/ml) directed against the amino acids 2–18 of the N-terminal domain of human MC-1R (33, 34) or a goat anti-MC-4R antibody directed against the carboxy terminus of MC-4R (1–3 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA). For triple staining cells were fixed with either paraformaldehyde and permeated as described above or paraformaldehyde only. Cells were blocked with 5% donkey/goat serum (for MC-1R) or 5% BSA/donkey serum (for MC-4R). Incubation with the anti-MC-R antibodies was performed as described above. After washing, cells were incubated for another hour with a monoclonal antibody against -smooth muscle actin (SMA) (Dunn, Asbach, Germany, 1:200). After washing, the slides were subsequently incubated for 30 min with secondary antibodies conjugated with fluorochromes, i.e. a donkey antirabbit antibody coupled to Texas Red (1:4000; Dianova, Hamburg, Germany) for the detection of bound MC-1R antibodies; a donkey antigoat antibody coupled to Texas Red (1:2400; Dianova) for the detection of bound MC-4R antibodies; or a goat antimouse antibody coupled to fluorescein isothiocyanate (FITC; Dako, Hamburg, Germany, 1:100) for the detection of bound SMA antibodies.
Nuclear staining was performed by incubating the cells with 4',6-diamido-2-phenylindole dihydrochloride (DAPI) at 100 ng/ml in Tris buffer (pH 7.4) for 1 h. The following procedure and antibodies were used for the detection of POMC peptide immunoreactivity. Cells were fixed with methanol for 30 min at –20 C. After blocking, cells were incubated for 1 h with an antibody against ED antibody (Oncogene Research; 1:100 to 101:500), anti-MSH antiserum (Peninsula, San Carlos, CA, 1:100–300), or two alternative anti-ACTH antibodies (BioCarta, Hamburg, Germany, 1 μg/ml; and Sigma, Deisenhofen, Germany, 1:100). Bound antibodies were detected by Texas Red-conjugated secondary antibodies as described above. Slides were finally mounted in Mowiol (Hoechst, Frankfurt, Germany) and imaged with an Axioscope 2 (Carl Zeiss). The following filter parameters were used: Texas Red, excitation, BP 530–585 nm, beamsplitter, FT 600 nm, emission, LP 615 nm; FITC, excitation, BP 450–490, beamsplitter, FT 510 nm, emission, LP 520 nm; DAPI, excitation, G 365 nm, beamsplitter, FT 395 nm, emission, LP 420. Images were taken by an Axiocam HR video camera and the Axiovision 3.0 software (Carl Zeiss). Negative controls were performed by either omitting the primary antibody, replacing it with preimmune serum (for MC-1R immunostaining), or incubating the cells with an isotype IgG control antibody (for MC-4R, ED and ACTH immunostaining) or by neutralization experiments (for ACTH and ED immunostaining) in which the primary antibody was preincubated with 10–6 M ACTH or ED (both from Bachem) for 1 h at room temperature in a small volume of PBS.
Determination of POMC-derived peptides
DPCs were seeded at a density of 500,000 cells into 6-cm diameter tissue culture dishes. On the following day, routine culture medium was switched to RPMI 1640 (PAA Laboratories, Linz, Austria) plus 2% FCS and supplements as indicated above. DPCs were maintained in culture for 3 consecutive days. Cell culture supernatants were then harvested, centrifuged and stored at –80 C. Alternatively, supernatants were concentrated with C18 columns (Waters, Midford, MA) after addition of protease inhibitors. Cell monolayers were either scraped into 250 μl PBS or Nonidet P-40 buffer supplemented with protease inhibitors. After sonication and centrifugation, supernatants were stored at –80 C. ACTH was measured by a commercially available immunometric assay from Diagnostic Products Corp. (Los Angeles, CA) with a sensitivity of less than 10 pg/ml. Cross-reactivities were as follows: ACTH 1–24, 1%; ACTH 18–39, 16.6%; MSH, 0.1%. MSH was measured with a radioimmune assay from Eurodiagnostica (Malm, Sweden) with a sensitivity of 5 pg/ml and no cross-reactivities against des-amino-MSH, ACTH 1–13, ACTH 1.24, ACTH 1–39, MSH, and MSH. ED was determined with a radioimmune assay from Nichols Institute Diagnostics (San Juan Capistrano, CA) with a sensitivity of 14 pg/ml for ED, and cross-reactivities were as follows: -lipotropin, 16%; ED, -lipotropin, and ED 1–27, 0.007%; and eucine enkephalin, methionine enkephalin, and ED, less than 0.001%. All measurements from more than three independent experiments were performed in duplicates and were analyzed by the Student’s t test.
Immunohistochemistry
Samples of normal scalp tissue from healthy individuals undergoing plastic surgery or patients undergoing routine surgery for therapeutic and diagnostic reasons (n > 5) were examined. Specimens were either cryofixed in liquid nitrogen or fixed in 7% buffered paraformaldehyde, dehydrated, embedded in paraffin, and mounted on Tissue-Tek (Mikrom, Walldorf, Germany). Paraffin sections were deparaffinized, epitope-demasked by microwave treatment, and quenched for endogenous peroxidase activity as described before (33). Sections were subsequently blocked with 2% BSA for 30 min at room temperature followed by incubation with an anti-MC-1R antibody (33, 34) at 1–2 μg/ml or with an anti-MC-4R antibody (Santa Cruz Biotechnology). Sections were washed and developed by the indirect immunoperoxidase technique using 3-amino-9-ethylcarbazole (Sigma) as a chromogen. For negative controls the primary antibody was replaced by preimmune serum or an isotype control IgG at the same concentration as the anti-MC-1R or anti-MC-4R antibody.
Results
Expression of POMC in human DPCs in culture
We first examined the RNA and protein expression of POMC, the precursor for melanocortins and endogenous opioids, in human DPCs in culture. RT-PCR analysis using established primer sets (27, 35, 36) revealed a single specific amplification product of the expected size (260 bp) in DPCs that comigrated with the positive control, i.e. normal human melanocytes (NHMs) (Fig. 1A). Contamination controls for DPCs were negative (Fig. 1A). To confirm the expression of POMC in human DPC at the protein level, we performed Western immunoblotting analysis using an antibody directed against the amino acids 18–39 of ACTH. This epitope is common in ACTH and POMC. Total cell lysates of DPCs expressed a single immunoreactive band of 32 kDa, which comigrated with the positive control (Fig. 1B). Lysates from SK-N-MC cells, a neuroblastoma cell line lacking POMC expression (37), did not produce any band (Fig. 1B). To confirm similar levels of protein loading between lysates from DPCs, NHMs, and SK-N-MC cells, the original blot was stripped and reprobed with an anti--tubulin antibody (Fig. 1B). In summary, these data show that human DPCs in vitro express the precursor hormone POMC at the mRNA and protein level.
Detection of immunoreactivity for POMC-derived peptides in human DPCs in culture
To check whether the expressed POMC in human DPCs may be processed into POMC-derived peptides, we first performed immunofluorescence studies using antibodies against ACTH, MSH, and ED. Immunoreactivity for all POMC peptides was detectable in DPCs, but ED immunostaining appeared to be most abundant. In approximately 40% of the cells, ED was detectable as a granular immunostaining confined to the cytoplasm and located mainly at the periphery of the cells (Fig. 1C). No specific staining was found in cells incubated with control rabbit IgG at the same concentration as the primary antibody (Fig. 1C). In addition, preincubation of the anti-ED antibody with ED resulted in complete neutralization of the staining (data not shown), suggesting that the observed immunostaining is due to ED or closely related ED-like peptides. In less than 20% of the cells, MSH immunoreactivity was also noted as a punctate immunostaining located within the cytoplasm (Fig. 1C). Omission of the primary antibody did not result in any immunostaining (Fig. 1C). ACTH immunoreactivity was also noted in the majority of DPCs, but this staining appeared to be nuclear (Fig. 1C) and could be reproduced by two different anti-ACTH antibodies. First, we used an affinity-purified monoclonal antibody directed against the amino acids 1–24 of ACTH, and, second, a polyclonal rabbit antiserum directed against the amino acids 18–39 of ACTH, both of which gave similar results. Control experiments in which the ACTH antibodies were omitted or replaced by control serum or an isotope control IgG did not result in any staining (data not shown). Moreover, preincubation of the used antibodies with ACTH abrogated the observed ACTH immunoreactivity (Fig. 1C), suggesting specificity of the staining.
To further investigate whether any of the detected POMC-derived peptides may be secreted by human DPCs in vitro, we determined the immunoreactive amounts of ACTH, MSH, and ED in culture media of these cells. Both crude and 50-fold concentrated culture supernatants were assayed. Among the tested POMC peptides, only ED was reproducibly detected in culture supernatants (extracellular fraction) as well as cell lysates (intracellular fraction). The detected amounts of ED were: intracellular, 9.63 ± 1.6 pg per 500,000 cells; and extracellular, 10.25 ± 1.06 pg per 500,000 cells. Our findings show that human DPCs in vitro express immunoreactivity for ACTH, MSH, and ED. Among the detected POMC-derived peptides, however, only ED appears to be secreted by DPCs in detectable amounts in our hands.
Studies on ASIP expression in human DPCs in culture
Expression of AP has been demonstrated within the dermal papilla of agouti mice in which it antagonizes the effect of melanocortins on eumelanin synthesis in hair follicle melanocytes (38). To clarify whether human DPCs express the human homolog of AP, ASIP, we performed RT-PCR and Western immunoblot analysis using an antibody that detects both AP and ASIP (31). cDNA from human adult testis was used as a positive for RT-PCR analysis (38). For Western immunoblotting, recombinant AP (85% homologous with ASIP at the amino acid level) as well as crude protein lysate from human testis was used as a positive control (38). In human DPC extracts, neither ASIP transcripts nor protein was detectable (Fig. 2, A and B). In contrast, human testis cDNA generated a specific amplification product of the expected size of 385 bp (29) (Fig. 2A). Western immunoblotting furthermore revealed the presence of an immunoreactive band of approximately 21.5 kDa in protein extracts from human testis (Fig. 2B), suggesting that ASIP protein is constitutively expressed in human adult testis.
Precursor proteases in human DPCs in culture
The generation of POMC-derived peptides by human DPCs suggested the expression of members of the subtilisin/kexin family of PCs, which cleave POMC into POMC-derived peptides. These enzymes are PC1, PC2 (together with its essential cofactor 7B2), PACE4, and furin convertase (FC) (3). In contrast to the previously reported detection of PC1 and PC2 in human epidermal melanocytes and dermal fibroblasts (27, 39), only transcripts for PC2 and 7B2 but not for PC1 were detected in human DPCs in culture in our hands (Fig. 3, A, C, and D). The PC2- and 7B2-related amplification products in DPCs were of the expected size of 299 (PC2) and 454 bp (7B2), whereas contamination controls were negative. The absence of a PC1-related amplification product in human DPCs was apparently not due to technical reasons because cDNA from NHM used as positive control clearly generated the expected PC-1 amplicon (Fig. 3A). To further search for other members of the PC family that may process POMC into POMC-derived peptides in human DPCs, we analyzed the expression of PACE4 and FC. We could not detect RNA expression for PACE4 whose transcripts were readily detectable in epidermal keratinocytes used as a positive control (40) (Fig. 3B). In contrast, we detected the expression of FC at the RNA level as demonstrated by amplification of the expected 101-bp product whose identity with FC was confirmed by DNA sequencing (data not shown) (Fig. 3E). These data demonstrate that at least two members of the family of precursor proteases, PC2 plus its essential cofactor 7B2, and FC are expressed in human DPCs in culture.
Detection of MC-1R and MC-4R in human DPCs in vitro and in situ
To investigate whether human DPCs are target cells for melanocortins, we first determined the RNA expression profile of all known MC-R subtypes by RT-PCR. DPC in vitro expressed transcripts for both MC-1R and MC-4R, whereas transcripts for MC-2R, MC-3R, and MC-5R were absent (Fig. 4A). The MC-1R and MC-4R amplification products from DPCs comigrated with the amplicons retrieved from the positive controls, which were NHM for MC-1R (416 bp) and genomic fibroblast DNA for MC-4R (566 bp) as reported previously (25, 26). Contamination controls were negative (Fig. 4A). MC-1R and MC-4R expression by human DPCs was subsequently supported by immunofluorescence studies. Paraformaldehyde-fixed DPCs exhibited a specific punctate staining pattern for both MC-1R and MC-4R (Fig. 4B). No such staining was detectable in cells incubated with preimmune serum or an isotype control used at the same concentration as the primary antibody (Fig. 4B). To further determine whether the detected MC-1R and MC-4R immunoreactivity is located in the plasma membrane and/or the cytoplasm, we performed experiments with permeated vs. nonpermeated cells. To this end, DPCs were also stained with an antibody against SMA and DAPI, the latter a nuclear marker. SMA is a well-established marker for myofibroblast transdifferentiation and is thus used for identification of DPCs. In our case, the anti-SMA antibody was used as a cytoplasmic indicator that would be visible only in permeated cells, i.e. those with paraformaldehyde fixation and detergent treatment, but not in cells with surface fixation, i.e. fixation with paraformaldehyde only. The results of these experiments are shown in Fig. 4C. In nonpermeated cells triple staining for MC-1R, SMA, and DAPI generated only a discrete punctate red labeling (due to MC-1R immunoreactivity) and a diffuse greenish cytoplasmic stain (due to nonspecific binding of the anti-SMA antibody) (Fig. 4C). MC-1R immunostaining became accentuated in permeated cells in which SMA appeared as the characteristic filament network (Fig. 4C). Similarly, MC-4R immunoreactivity was more abundant in permeated than nonpermeated cells (Fig. 4C). The data indicate that immunoreactivity for both MC-1R and MC-4R in human DPCs is located in the plasma membrane as well as the cytoplasm, the latter likely to correspond to newly synthesized receptors being moved to the plasma membrane via endoplasmic reticulum, Golgi apparatus, and vesicular pathways.
To finally learn about the expression of MC-1R and MC-4R in DPCs in situ, we performed immunohistochemical studies on tissue sections of normal adult human scalp skin. MC-1R and MC-4R immunoreactivity was detectable within the cytoplasm of fibroblastic cells of the dermal papilla (Fig. 4D). No immunostaining was observed in negative controls in which the primary antibody was replaced by preimmune serum or the isotype control (Fig. 4D). These findings corroborate the previously detected MC-1R and MC-4R expression in human DPCs in culture and suggest that these cells are also targets for MSH in vivo.
Evidence for functionally active MC-Rs in human DPCs in vitro
We next assessed the biological relevance of MC-Rs expressed by human DPCs. We initially focused on the effect of MSH on intracellular cAMP because engagement of all types of MC-Rs with their cognate ligands results in immediate activation of adenylyl cyclase. Based on previous findings that showed a significant dose-dependent cAMP response of human dermal fibroblasts 20 min after MSH treatment (25), we also decided to use this time point for our studies with human DPCs. Multiple independent experiments (n > 6) with cells from different donors revealed that only MSH doses of 10–6 M elicited a robust increase in intracellular cAMP (Fig. 5A). As expected, the artificial cAMP inducer forskolin (1 μM) used as positive control also increased intracellular cAMP (Fig. 5A). Neither changes in the amount of FCS during the deprivation phase of the cells or preincubation with IBMX for 1 h nor modified time points of MSH treatment resulted in significant increases in intracellular cAMP at MSH concentrations beyond 10–6 M (data not shown). However, because previous studies had provided compelling evidence that the human MC-1R is activated in nanomolar amounts of MSH (5, 6), we wanted to further specify the MSH-mediated cAMP response in human DPCs. Therefore, cells were preincubated with 10–6 M of a synthetic peptide corresponding to the amino acids 87–132 of the carboxy-terminal domain of ASIP followed by stimulation with MSH at the same concentration. Previously it has been shown that cysteine-rich C-terminal ASIP fragments are as potent as full-length ASIP (41). As depicted in Fig. 5B, the MC-1R/MC-4R antagonistic peptide largely abrogated the MSH-mediated cAMP response in human DPCs. These findings strongly suggest that the observed cAMP response by MSH in human DPCs is mediated via activation of MC-1R and/or MC-4R, both of which are expressed in these cells as presented above.
To further assess the functionality of the MC-Rs expressed in human DPCs by yet another signal transduction pathway known to be activated by MSH, we investigated the changes in intracellular calcium, an event crucially involved in elicitation of the biological effect of MSH (42). Recently rapid and acute increase in intracellular calcium has also been described in normal human keratinocytes and HaCaT cells after stimulation with MSH and ACTH peptides (43). In accordance with the detection of calcium spikes induced by MSH in keratinocytes, treatment of human DPCs with MSH resulted in a significant intracellular calcium spike within 10 sec. Both MSH doses of 10–6 M (data not shown) but also 10–8 M consistently evoked this response, whereas MSH at 10–10 M did not have any effect (Fig. 5C). The calcium spikes induced by MSH were transient and returned to baseline levels by 60 sec. As expected, treatment of the cells with inomycin (10–4 M) used as positive control also led to a robust calcium spike in human DPCs (Fig. 5C). These findings show that in addition to the cAMP pathway, the calcium signaling pathway is activated by MSH in human DPCs.
Because MSH has been reported to exhibit immunoregulatory activities (44, 45, 46) and aberrant expression of ICAM-1 has been implicated in the pathogenesis of alopecia areata (47, 48, 49), an inflammatory, nondestructive type of hair loss, we finally investigated whether MSH can modulate basal and cytokine-induced ICAM-1 expression in human DPCs in vitro. IFN, a prototypical inducer of ICAM-1 (50), strongly induced mRNA expression of ICAM-1 as shown by RT-PCR (Fig. 5D). MSH alone did not have any modulatory activity on basal expression of ICAM-1. When coincubated with IFN, MSH at 10–6 M significantly suppressed the inductive effect of IFN on ICAM-1 expression. In accordance with our data on the cAMP induction by MSH in human DPCs, concentrations of 10–8 M failed to suppress the inductive effect of IFN on ICAM-1 (data not shown).
Collectively, these findings indicate that the MC-Rs expressed by human DPCs in vitro are functionally active. MSH triggers at least two classical signal transduction pathways in these cells, the cAMP and calcium pathway. In addition, MSH exerts immunoregulatory effects in these cells such as suppression of adhesion molecule expression induced by a prototypical proinflammatory cytokine.
Discussion
In this study we report the detection of several components of the POMC system in human DPCs, i.e. POMC, transcripts for distinct members of the PC family of precursor proteases, immunoreactivity for POMC-derived peptides, and the presence of receptors for MSH.
Most importantly, the receptors for MSH, MC-1R, and MC-4R appear to be functionally active in human DPCs because MSH elicits an increase in intracellular cAMP and calcium as well as down-regulates the expression of ICAM-1 induced by IFN. Concomitant expression of different MC-Rs has been described in several cutaneous cell types. Human epidermal melanocytes were shown to express not only MC-1R (51) but also MC-2R at the RNA level (52). Likewise, expression of MC-1R and MC-2R was reported in human keratinocytes (53, 54). These findings are supported by the detection of MC-2R at the RNA level in whole human and murine skin (10, 52). In addition, dual expression of MC-2R and MC-5R has been reported in the murine 3T3-L1 adipocyte cell line (55). We are unaware of any report showing expression of MC-4R in human skin. Expression of MC-4R is primarily found in the brain. However, MC-4R has recently been reported to occur in a number of extraneural tissues including the skin in the fetal rat, suggesting a role of MC-4R beyond appetite, weight control, and regulation of linear growth (56). The biological function of dual expression of different MC-R subtypes is enigmatic. The human MC-1R has the highest affinity for MSH and an almost equal affinity for ACTH, whereas the MC-4R is equipotently activated by both melanocortins (6). It is possible that the relative level of MC-Rs expressed dictates the biological response of a cell to the ligand due to MC-R signal transduction heterogeneity. It has been reported that albeit both MC-3R and MC-4R activate adenylyl cyclase, activation of the MAPK cascade is induced only by MC-4R (57). Because the used ASIP peptide in our studies was antagonistic to both MC-1R and MC-4R, further research is needed to dissect the signaling pathways attributable to each MC-R subtype expressed by human DPCs. Future studies will also have to clarify why the cAMP response (as well as the immunoregulatory effect) of MSH is induced only at 10–6 M. The melanotropic response of MSH in normal human melanocytes is typically induced at nanomolar doses as predicted by the MC-1R binding affinity (51). Ligand binding studies should help to determine the actual number of MC-Rs being expressed on the surface of human DPCs.
Regarding the immunoregulatory effect of MSH on the expression of ICAM-1 in human DPCs, our findings extend those of others who also reported regulatory activities of MSH on the expression of adhesion molecules. Accordingly, MSH was shown to suppress ICAM-1 expression induced by TNF in normal and transformed human melanocytes (58). MSH also suppressed lipopolysaccharide-induced expression of ICAM-1, vascular adhesion molecule-1, and E-selectin by dermal endothelial cells in a mouse model of experimentally induced vasculitis (59). Ito et al. (60) recently demonstrated that MSH can suppress ectopically induced MHC class I and II expression in organ-cultured anagen human hair bulbs. Typically the dermatohistopathologic picture of the hair follicles in patients with alopecia areata consists of peribulbar T lymphocytes forming a bee swarm-like infiltrate. In light of the collapse of the immune privilege in alopecia areata leading to aberrant adhesion molecule expression (61) and the presence of functional MC-Rs in DPCs, it will be worthwhile to examine the expression and functionality of MC-Rs in DPCs of patients with this disorder. In addition, it will be interesting to examine the expression of POMC, POMC-derived peptides, and PCs in this condition as already suggested by others (62).
Unexpectedly we have been unable to detect immunoreactive amounts of ACTH and MSH by means of immunometric assays in human DPCs in culture, although these cells expressed immunoreactivity for both of these POMC peptides as shown by immunofluorescence studies. This is in contrast to epidermal melanocytes and keratinocytes (36), human dermal fibroblasts (27), and microvascular endothelial cells (63), all of which release immunoreactive amounts of melanocortins into the culture media. Biochemical studies using reversed-phase HPLC as well as liquid chromatography-mass spectrometry confirmed the presence of the above POMC peptides in cultured human melanocytes, melanoma, and squamous cell carcinoma cells as well as normal and diseased human skin (64, 65, 66). At present, we cannot exclude that MSH and ACTH produced by human DPCs are rapidly degraded in vitro and thus escape from detection. Interestingly, the ACTH immunoreactivity observed in human DPCs appeared to be nuclear and could be generated by two antibodies directed against two different epitopes of ACTH. Because preincubation with ACTH completely abrogated the immunostaining, it is most likely that the used antibodies detect ACTH or closely related ACTH peptides. Of note, nuclear localization has been reported for a number of growth factors and hormones including pro-CRH (67). However, MSH immunoreactivity in DPCs was found within the cytoplasm, which is puzzling in light of the nuclear staining of ACTH and the known processing of POMC. Therefore, additional studies such as immunofluorescence analysis with different fixation methods and use of organelle markers, subcellular fractionation with biochemical analysis of ACTH, and immune electron microscopy should define the exact localization of ACTH in human DPC. In contrast to MSH and ACTH, we were able to detect ED immunoreactivity and immunoreactive amounts of this POMC peptide by radioimmune assay in the culture media of ACTH and immune human DPCs. It needs to be determined whether human DPCs indeed secrete bioactive amounts of this neuropeptide.
Answering this question is necessary because ED is posttranslationally modified by N-acetylation to yield inactive forms of the peptide (68, 69). Given the detected expression of PC2, 7B2, and FC in human DPCs and the enzymatic activity of these enzymes against POMC (3), it is conceivable that these enzymes may be involved in generation of ED. However, during revision of this manuscript, Kauser et al. (70) reported on the expression of the POMC system in the human hair follicle. These authors detected the expression of both PC1 and PC2 in cultured DPCs and demonstrated immunoreactivity for both enzymes in the human dermal papilla in situ. In murine skin, immunoreactivity for PC1 and PC2 was present in epidermal keratinocytes and the sebaceous unit as well as some adjacent cells (13). Both PC1 and PC2 displayed a differential temporal and spatial pattern of immunoreactivity. The reason for the discrepancy between our findings and those of others (70) is unclear but may be related to donor variability and/or differences in the cell culture conditions.
In summary, we have presented herein evidence for functionally active receptors for MSH in human DPCs. In addition, we have detected several components of the POMC system in these cells. Our data as well as those from others (70) suggest close interconnections via the POMC system among the cellular components of the hair follicle as depicted in Fig. 6. Via MC-1R and MC-4R, MSH (and possibly other melanocortins) may regulate inflammatory reactions such as expression of ICAM-1 in DPCs, thereby modulating the recruitment of T lymphocytes into the dermal papilla. On the other hand, POMC-derived peptides presumably released by DPCs may act as paracrine regulators of hair follicle epithelia and melanocytes. For example, MSH, ACTH, and ED released by DPCs may stimulate melanogenesis because human hair follicle melanocytes express not only MC-1R but also the μ-opioid receptor (70, 71). Because interfollicular keratinocytes express μ-opioid receptor (72) as well and respond to ED with increased cytokeratin 16 expression (73), it is possible that ED released from DPCs may regulate keratinization of the hair follicle in analogy. In light of the diversity of biological actions of MSH such as regulation of extracellular matrix composition and apoptosis (25, 74), future studies can be expected to add novel insight into our understanding of the POMC system and the hair follicle.
Acknowledgments
The authors acknowledge the expert technical assistance of Ilka Wolff.
Footnotes
This work was supported by a research grant from the Deutsche Forschungsgemeinschaft (DFG, 1075/5-1, to M.B.) and a research grant on alopecia from Pfizer.
Abbreviations: AP, Agouti protein; ASIP, agouti signaling protein; DAPI, 4',6-diamido-2-phenylindole dihydrochloride; DPC, dermal papilla cell; ED, endorphin; FC, furin convertase; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBRS, HEPES-buffered Ringer solution; IBMX, isobutyl methylxanthine; ICAM, intercellular adhesion molecule; IFN, interferon; MC-R, melanocortin receptor; NHM, normal human melanocyte; PACE4, paired basic amino acid cleaving enzyme 4 gene; PC, prohormone convertase; POMC, proopiomelanocortin; SMA, -smooth muscle actin.
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Department of Pediatrics and Genetics and the Howard Hughes Medical Institute (G.S.B.), Stanford University School of Medicine, Stanford, California 94305
Department of Dermatology (A.V., K.S., U.B.-P.), Center for Applied Cutaneous Physiology, University Hospital Charité, D-10117 Berlin, Germany
Abstract
Proopiomelanocortin (POMC)-derived peptides and their receptors have been identified in many peripheral organs including the skin in which they exert a diversity of biological actions. We investigated the expression and potential role of the POMC system in human dermal papilla cells (DPCs), a specialized cutaneous mesenchymal cell type regulating hair follicle activity. In culture, these cells expressed POMC and displayed immunoreactivity for ACTH, MSH, and -endorphin. Among the prohormone convertases (PCs) tested, only PC2, its chaperone 7B2, and furin convertase but not PC1 and paired basic amino acid cleaving enzyme 4 gene were detected. Human DPCs in vitro expressed both the melanocortin-1 receptor (MC-1R) and MC-4R, and immunoreactivity for these receptors was also present in cells of the human dermal papilla in situ. In contrast to the dermal papilla of agouti mice, agouti signaling protein, a natural and highly selective MC-1R and MC-4R antagonist, was undetectable in human DPCs. The MC-Rs detected in human DPCs were functionally active because MSH increased intracellular cAMP and calcium. Preincubation of the cells with a synthetic peptide corresponding to the C-terminal domain of agouti signaling protein abrogated cAMP induction by MSH. Furthermore, MSH was capable of antagonizing the expression of intercellular adhesion molecule-1 induced by the proinflammatory cytokine interferon-. Our data suggest a regulatory function of MSH within the dermal papilla whose disruption may lead to deregulation of immune and inflammatory responses of the hair follicle, thereby possibly contributing to the development of inflammatory forms of alopecia.
Introduction
THE PROOPIOMELANOCORTIN (POMC) (1) system is an evolutionary conserved regulatory module that is expressed in the brain but also in many peripheral tissues including the skin (1, 2). In the skin the POMC system is crucially involved in a diversity of physiologic processes such as pigmentation, inflammation, lipogenesis, and exocrine gland activity. Because the skin expresses all regulatory components of the POMC system and responds to stressful stimuli with induction of this system, it has been suggested that an equivalent of the hypothalamic-pituitary-adrenal axis is operational in the skin (1).
POMC contains within its structure the amino acid sequences of several hormonal products including the melanocortins, which are ACTH, MSH, MSH, and MSH as well as the endogenous opioids -, - and -endorphin (ED). Generation of these hormones from POMC requires the action of at least two precursor or prohormone convertases (PCs) belonging to the family of serine proteases. Processing of POMC at dibasic residues is mediated by either PC1 alone (resulting in ACTH and -lipotropin) or a combination of PC1 and PC2 plus the PC2-specific binding protein 7B2 yielding MSH and ED (3). There is recent evidence from in vitro and in vivo data that additional PCs such as paired basic amino acid cleaving enzyme 4 gene (PACE4) or furin convertase may also process POMC (3, 4). Whereas ED elicits its biological effects via binding to opioid receptors, the melanocortins mediate their biological actions by binding to melanocortin receptors (MC-Rs). Five MC-Rs (MC-1R to MC-5R) have been cloned belonging to the superfamily of G protein-coupled receptors with seven transmembrane domains (5). They differ in their relative binding affinity to ACTH and the various MSH peptides (5, 6).
With regard to the hair follicle, it is known for many years that MSH affects the coat color of mice (7). Subsequent studies provided accumulating evidence that many components of the POMC system including the upstream regulators, CRH and its receptor, are expressed in the pilosebaceous unit of mouse skin (8, 9, 10, 11, 12, 13). In these studies POMC and POMC-derived peptides exhibited differential spatial and temporal expression in the pilosebaceous unit and the hair follicle, i.e. expression varied, depending on the hair cycle. In mice, the functional significance of melanocortins in coat color regulation is highlighted by the fact that animals with a signaling-deficient MC-1R (recessive yellow mutation) display yellow pigment due to the shift of eumelanin toward pheomelanin synthesis (14). On the other hand, mice ubiquitously expressing agouti protein (AP), a natural antagonist of MC-1R and MC-4R, have a completely yellow fur (15). It was also reported that in vivo administration of certain melanocortins to animals can modulate hair growth. In the mink, anagen hair growth, i.e. the phase of the hair cycle during which synthesis of hair takes place, can be induced by intracutaneous injection of ACTH but not by MSH (16). When injected into murine telogen skin (the quiescent phase of the hair cycle), ACTH has a similar anagen-inducing effect (17). Immunohistochemical studies on the back skin of neonatal C57BL/6 mice and adolescent C57BL/6 mice with depilation-induced hair cycle further showed that MSH and MC-1R immunoreactivity are differentially expressed in various cells of the hair follicle during hair follicle morphogenesis and cycling, suggesting a role of melanocortins beyond hair follicle pigmentation (18).
In man, genetic studies have corroborated the functional role of the MC-1R for hair color. It was shown that northern European populations with red hair and sun sensitivity display common MC-1R alleles including the Arg151Cys, Arg160Trp, and Asp294His (19). Regarding the ligands for MC-1R, it was further demonstrated that POMC mutations leading to trace amounts of POMC-derived peptides or complete absence of ACTH, MSH, and ED result in red hair (20). Finally, it is also known that therapeutic administration of ACTH can lead to hypertrichosis and hirsutism (21).
Although these findings suggest that the human hair follicle is a target site for melanocortins, expression and function of the POMC system in this adnexal structure of the skin remained incompletely characterized until recently. In this report we investigated whether human dermal papilla cells (DPCs), a unique myofibroblast cell type involved in hair follicle activity, express components of the POMC system. Our studies revealed that various elements of the POMC system are expressed in DPCs established from human skin. The expressed MC-Rs are functionally active because prototypical responses such as cAMP and calcium signaling can be elicited by MSH. Finally, our studies on the immunoregulatory action of MSH in human DPCs emphasize a functional aspect that may be of particular relevance in the pathogenesis of inflammatory forms of alopecia and/or its future therapy.
Materials and Methods
Establishment of human DPC cultures
Isolation and culture of human DPCs was performed as reported previously (22). The Institutional Review Board of the University Hospital Charité, Berlin, had previously approved all of the described studies. All experiments adhered to the Declaration of Helsinki guidelines. DPC cultures were established from normal scalp tissue of healthy patients undergoing plastic surgery for face lifts or from the margins of unaffected scalp skin after surgical excision of benign cutaneous lesions. Scalp skin was collected in DMEM with 20% fetal calf serum (FCS), 100 U/ml penicillin/streptomycin, 0.4 mM [SCAP];l-glutamine, and 50 ng/ml amphotericin B (all from Biochrom, Berlin, Germany). Complete anagen follicles were microdissected under a stereomicroscope (47 50 20; Carl Zeiss, Jena, Germany) according to the technique of Jahoda and Oliver (23) as modified by Reynolds and Jahoda (24). Each dermal papilla was cut across the stalk region, transferred to a 35-mm culture dish, and incubated in a humidified atmosphere with 5% CO2 at 37 C. Confluent DPC cultures were subcultured after 8 wk and propagated in the culture medium as described above. DPC cultures were used at passage numbers 2–4. Cell cultures were usually discontinued after five passages.
RNA extraction, RT-PCR, and sequencing
Total RNA was prepared using the RNeasy kit (QIAGEN, Santa Clarita, CA). After DNA digestion with DNAse (Promega, Madison, WI), 2 μg of total RNA were reverse transcribed with 15 U AMV-RT (Promega) and amplified with primers against MC-1R to MC-5R, PC1, PC2, 7B2, and POMC under identical conditions as reported (25, 26, 27). PACE4 was amplified with primer sets established by others (28). All amplification products were of the expected size. For the detection of the 111-bp amplification product of furin convertase, the following primers were used: forward, 5'-TGC TGG TCT TCG TCA CTG TC-3' and reverse, 5'-TTG TAG GAG ATG AGG CCA CGG-3'. The amplification protocol was 1 x 94 C for 5 min; 60 C for 1 min; 72 C for 2 min; 35 x 94 C for 45 sec; 60 C for 2 min; 72 C for 1 min; 1 x 94 C for 45 sec; 60 C for 1 min; and 72 C for 10 min. To confirm the identity of the latter product, the respective band was extracted from the 2% agarose gel using an extraction kit from QIAGEN. The extracted DNA was cloned into a pGEM-T easy vector (Promega) and sequenced (4base lab GmbH, Reutlingen, Germany). Expression of agouti signaling protein (ASIP) was performed as reported previously with slight modifications (29). The primers were: forward, 5'-ATGGATGTCACCCGCTTACT-5' and reverse, 5'-GCGGAAGAAGCGGCACTGG-3', generating a 385-bp amplicon. The amplification protocol was 1 x 94 C for 5 min; 60 C for 1 min; 58 C for 2 min, 72 C for 2 min; 31 x 94 C for 45 sec; 58 C for 1 min; 72 C for 2 min; 1 x 94 C for 45 sec; 58 C for 2 min; and 72 C for 10 min. As a positive control for ASIP, microdissected normal human testis derived from routine surgical procedures (kindly provided by Dr. C. Poremba, Department of Pathology, University of Düsseldorf, Düsseldorf, Germany) was used.
Quantitative real-time PCR
For RNA expression studies of intercellular adhesion molecule (ICAM)-1, DPCs were deprived for 24 h in 1% FCS followed by stimulation with 100 ng/ml interferon (IFN)- alone or in combination with 10–6 M -MSH (Bachem, Bubendorf, Switzerland) for 4 h. In some experiments cells were also stimulated with 10–8 M MSH. Quantification of mRNA levels was carried out by real-time fluorescence detection as previously reported (25, 30). Primers and probes for ICAM-1 and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from PE Applied Biosystems (Foster City, CA) (GAPDH: Hs99999905; ICAM-1: HS00164932-M1). cDNA was amplified by PCR in the ABI Prism 7700 sequence detector (PE Applied Biosystems). 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, Darmstadt, Germany) 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 normal human dermal fibroblasts. The relative amounts of gene expression were calculated by using GAPDH as a standard. Each PCR analysis was performed in triplicates from a total of three independent experiments. Data were expressed as means ± SD and analyzed by the Student’s t test.
Determination of cAMP
DPC (2 x 104 cells/well) were seeded into 96-well tissue culture plates in regular culture medium. On the next day, cells were thoroughly washed and switched to RPMI 1640 without FCS. Cells were subsequently deprived from FCS for 48 h followed by stimulation with MSH for 20 min in the presence of 0.1 mM isobutyl methylxanthine (IBMX). In some experiments IBMX was also preincubated for 1 h before stimulation with MSH. In another set of experiments, DPCs were stimulated with MSH for 10 and 40 min. For competitive blocking experiments, a synthetic peptide containing the amino acids 87–132 of the C-terminal domain of ASIP (Phoenix Pharmaceuticals, Belmont, CA) was incubated with the cells at 10–6 M 15 min before stimulation with MSH at the same concentration. In all experiments forskolin at 0.1–1 μM served as a positive control. Cells were subsequently lysed and cAMP was determined by a specific enzyme immunoassay (Amersham Pharmacia Biotech, Freiburg, Germany). Triplicate wells were used for each treatment and statistical analysis was performed using the Student’s t test.
Calcium measurements
For detection of intracellular calcium levels, DPCs were trypsinized, plated on glass coverslips, and grown in regular culture medium for 1–2 d. Subconfluent cell monolayers were then deprived from FCS for 48 h. Cells were incubated with 4.5 μmol/liter of the fluorescent dye FURA2-AM in HEPES-buffered Ringer solution (HBRS) containing (in millimoles per liter): 140 NaCl, 5 KCl, 1 MgCl, 1 CaCl2, 5 glucose, 10 HEPES adjusted to pH 7.4 for 15 min at 37 C. Then they were washed three times with FURA2-free HBRS. Coverslips with loaded cells were placed in a temperature-controlled (37 C) perfusion chamber and mounted on the stage of inverted microscope (Diaphot 300; Nikon, Tokyo, Japan). About six cells were focused. Excitation wavelength alternated between 340 and 380 nm, and emitted fluorescence was monitored at 535 nm by a microscope photometer (hardware and software for controlling and achieving alternating excitation and data acquisition obtained by Photon Technology International, Hamburg, Germany). The tracings were corrected in each experiment for autofluorescence before calculating the 340:380 ratio. During the experiment, cells were continuously superfused with HBRS. For calcium measurements the perfusion media were supplemented with MSH at various concentrations ranging from 10–6 to 10–10 M. As positive control, inonomycin at 10–4 M was used.
Western immunoblotting
Total cell lysates were prepared by scraping the cells into lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM ethylenglycol-bis(-aminoethylether)-N,N,N'N'-tetraacetic acid and 0.01% NaN2 plus freshly added proteinase inhibitors (10 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). After sonication and centrifugation, supernatants were analyzed for protein content using the modified Bradford assay (Bio-Rad Laboratories, Richmond, CA). An equal volume of 2x sodium dodecyl sulfate loading buffer was added and the samples were boiled at 95 C for 5 min. Equal protein amounts were separated by denaturing SDS-PAGE using 4–12% gradient gels from NuPAGE (Invitrogen, Carlsbad, CA) followed by immunoblotting. The polyvinylidene difluoride membranes were blocked overnight with 10% BSA. For the detection of immunoreactive POMC, an anti-ACTH antibody (Sigma, St. Louis, MO) directed against the amino acids 18–39 of ACTH (1:1000). For the detection of ASIP, sample preparation and SDS-PAGE were carried out under nonreducing conditions. Generation of the rabbit polyclonal anti-AP antibody directed against the full-length (His23-Cys131) of AP is described elsewhere (31). The used anti-AP antibody detects 1 ng/lane of the immunogen and cross-reacts with ASIP (31). Protein lysate from human testis derived from routine surgical procedures (kindly provided by Dr. C. Poremba, Department of Pathology, University of Düsseldorf, Düsseldorf, Germany) served as a positive control for ASIP. Membranes were incubated with primary antibodies overnight, washed, and incubated for 20 min with a horseradish-peroxidase-conjugated secondary antibody (1:10,000; Amersham Life Science, Freiburg, Germany). Antigen-antibody complexes were visualized with the enhanced chemiluminescence kit (Amersham). To confirm identical loading of lysate proteins the original blot was stripped as reported before (32) followed by reblocking and incubation with an anti--tubulin antibody (1:1000; Oncogene Research Products, San Diego, CA).
Immunofluorescence studies
DPCs were seeded into 8-well Lab-Tek Permanox chamber slides (Nalge Nunc International, Naperville, IL) at a density of 10,000 cells/well. On the following day, cells were either fixed with 4% paraformaldehyde for 20 min at room temperature (for the detection of plasma membrane MC-1R and MC-4R immunoreactivity) or subsequently permeated with 0.3% Triton X-100 for 10 min (for the detection of cytoplasmic MC-1R and MC-4R immunoreactivity). Nonspecific binding was blocked with 5% donkey serum for 1 h. Cells were then incubated for 1 h with either a rabbit anti-MC-1R antibody (at 1–5 μg/ml) directed against the amino acids 2–18 of the N-terminal domain of human MC-1R (33, 34) or a goat anti-MC-4R antibody directed against the carboxy terminus of MC-4R (1–3 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA). For triple staining cells were fixed with either paraformaldehyde and permeated as described above or paraformaldehyde only. Cells were blocked with 5% donkey/goat serum (for MC-1R) or 5% BSA/donkey serum (for MC-4R). Incubation with the anti-MC-R antibodies was performed as described above. After washing, cells were incubated for another hour with a monoclonal antibody against -smooth muscle actin (SMA) (Dunn, Asbach, Germany, 1:200). After washing, the slides were subsequently incubated for 30 min with secondary antibodies conjugated with fluorochromes, i.e. a donkey antirabbit antibody coupled to Texas Red (1:4000; Dianova, Hamburg, Germany) for the detection of bound MC-1R antibodies; a donkey antigoat antibody coupled to Texas Red (1:2400; Dianova) for the detection of bound MC-4R antibodies; or a goat antimouse antibody coupled to fluorescein isothiocyanate (FITC; Dako, Hamburg, Germany, 1:100) for the detection of bound SMA antibodies.
Nuclear staining was performed by incubating the cells with 4',6-diamido-2-phenylindole dihydrochloride (DAPI) at 100 ng/ml in Tris buffer (pH 7.4) for 1 h. The following procedure and antibodies were used for the detection of POMC peptide immunoreactivity. Cells were fixed with methanol for 30 min at –20 C. After blocking, cells were incubated for 1 h with an antibody against ED antibody (Oncogene Research; 1:100 to 101:500), anti-MSH antiserum (Peninsula, San Carlos, CA, 1:100–300), or two alternative anti-ACTH antibodies (BioCarta, Hamburg, Germany, 1 μg/ml; and Sigma, Deisenhofen, Germany, 1:100). Bound antibodies were detected by Texas Red-conjugated secondary antibodies as described above. Slides were finally mounted in Mowiol (Hoechst, Frankfurt, Germany) and imaged with an Axioscope 2 (Carl Zeiss). The following filter parameters were used: Texas Red, excitation, BP 530–585 nm, beamsplitter, FT 600 nm, emission, LP 615 nm; FITC, excitation, BP 450–490, beamsplitter, FT 510 nm, emission, LP 520 nm; DAPI, excitation, G 365 nm, beamsplitter, FT 395 nm, emission, LP 420. Images were taken by an Axiocam HR video camera and the Axiovision 3.0 software (Carl Zeiss). Negative controls were performed by either omitting the primary antibody, replacing it with preimmune serum (for MC-1R immunostaining), or incubating the cells with an isotype IgG control antibody (for MC-4R, ED and ACTH immunostaining) or by neutralization experiments (for ACTH and ED immunostaining) in which the primary antibody was preincubated with 10–6 M ACTH or ED (both from Bachem) for 1 h at room temperature in a small volume of PBS.
Determination of POMC-derived peptides
DPCs were seeded at a density of 500,000 cells into 6-cm diameter tissue culture dishes. On the following day, routine culture medium was switched to RPMI 1640 (PAA Laboratories, Linz, Austria) plus 2% FCS and supplements as indicated above. DPCs were maintained in culture for 3 consecutive days. Cell culture supernatants were then harvested, centrifuged and stored at –80 C. Alternatively, supernatants were concentrated with C18 columns (Waters, Midford, MA) after addition of protease inhibitors. Cell monolayers were either scraped into 250 μl PBS or Nonidet P-40 buffer supplemented with protease inhibitors. After sonication and centrifugation, supernatants were stored at –80 C. ACTH was measured by a commercially available immunometric assay from Diagnostic Products Corp. (Los Angeles, CA) with a sensitivity of less than 10 pg/ml. Cross-reactivities were as follows: ACTH 1–24, 1%; ACTH 18–39, 16.6%; MSH, 0.1%. MSH was measured with a radioimmune assay from Eurodiagnostica (Malm, Sweden) with a sensitivity of 5 pg/ml and no cross-reactivities against des-amino-MSH, ACTH 1–13, ACTH 1.24, ACTH 1–39, MSH, and MSH. ED was determined with a radioimmune assay from Nichols Institute Diagnostics (San Juan Capistrano, CA) with a sensitivity of 14 pg/ml for ED, and cross-reactivities were as follows: -lipotropin, 16%; ED, -lipotropin, and ED 1–27, 0.007%; and eucine enkephalin, methionine enkephalin, and ED, less than 0.001%. All measurements from more than three independent experiments were performed in duplicates and were analyzed by the Student’s t test.
Immunohistochemistry
Samples of normal scalp tissue from healthy individuals undergoing plastic surgery or patients undergoing routine surgery for therapeutic and diagnostic reasons (n > 5) were examined. Specimens were either cryofixed in liquid nitrogen or fixed in 7% buffered paraformaldehyde, dehydrated, embedded in paraffin, and mounted on Tissue-Tek (Mikrom, Walldorf, Germany). Paraffin sections were deparaffinized, epitope-demasked by microwave treatment, and quenched for endogenous peroxidase activity as described before (33). Sections were subsequently blocked with 2% BSA for 30 min at room temperature followed by incubation with an anti-MC-1R antibody (33, 34) at 1–2 μg/ml or with an anti-MC-4R antibody (Santa Cruz Biotechnology). Sections were washed and developed by the indirect immunoperoxidase technique using 3-amino-9-ethylcarbazole (Sigma) as a chromogen. For negative controls the primary antibody was replaced by preimmune serum or an isotype control IgG at the same concentration as the anti-MC-1R or anti-MC-4R antibody.
Results
Expression of POMC in human DPCs in culture
We first examined the RNA and protein expression of POMC, the precursor for melanocortins and endogenous opioids, in human DPCs in culture. RT-PCR analysis using established primer sets (27, 35, 36) revealed a single specific amplification product of the expected size (260 bp) in DPCs that comigrated with the positive control, i.e. normal human melanocytes (NHMs) (Fig. 1A). Contamination controls for DPCs were negative (Fig. 1A). To confirm the expression of POMC in human DPC at the protein level, we performed Western immunoblotting analysis using an antibody directed against the amino acids 18–39 of ACTH. This epitope is common in ACTH and POMC. Total cell lysates of DPCs expressed a single immunoreactive band of 32 kDa, which comigrated with the positive control (Fig. 1B). Lysates from SK-N-MC cells, a neuroblastoma cell line lacking POMC expression (37), did not produce any band (Fig. 1B). To confirm similar levels of protein loading between lysates from DPCs, NHMs, and SK-N-MC cells, the original blot was stripped and reprobed with an anti--tubulin antibody (Fig. 1B). In summary, these data show that human DPCs in vitro express the precursor hormone POMC at the mRNA and protein level.
Detection of immunoreactivity for POMC-derived peptides in human DPCs in culture
To check whether the expressed POMC in human DPCs may be processed into POMC-derived peptides, we first performed immunofluorescence studies using antibodies against ACTH, MSH, and ED. Immunoreactivity for all POMC peptides was detectable in DPCs, but ED immunostaining appeared to be most abundant. In approximately 40% of the cells, ED was detectable as a granular immunostaining confined to the cytoplasm and located mainly at the periphery of the cells (Fig. 1C). No specific staining was found in cells incubated with control rabbit IgG at the same concentration as the primary antibody (Fig. 1C). In addition, preincubation of the anti-ED antibody with ED resulted in complete neutralization of the staining (data not shown), suggesting that the observed immunostaining is due to ED or closely related ED-like peptides. In less than 20% of the cells, MSH immunoreactivity was also noted as a punctate immunostaining located within the cytoplasm (Fig. 1C). Omission of the primary antibody did not result in any immunostaining (Fig. 1C). ACTH immunoreactivity was also noted in the majority of DPCs, but this staining appeared to be nuclear (Fig. 1C) and could be reproduced by two different anti-ACTH antibodies. First, we used an affinity-purified monoclonal antibody directed against the amino acids 1–24 of ACTH, and, second, a polyclonal rabbit antiserum directed against the amino acids 18–39 of ACTH, both of which gave similar results. Control experiments in which the ACTH antibodies were omitted or replaced by control serum or an isotope control IgG did not result in any staining (data not shown). Moreover, preincubation of the used antibodies with ACTH abrogated the observed ACTH immunoreactivity (Fig. 1C), suggesting specificity of the staining.
To further investigate whether any of the detected POMC-derived peptides may be secreted by human DPCs in vitro, we determined the immunoreactive amounts of ACTH, MSH, and ED in culture media of these cells. Both crude and 50-fold concentrated culture supernatants were assayed. Among the tested POMC peptides, only ED was reproducibly detected in culture supernatants (extracellular fraction) as well as cell lysates (intracellular fraction). The detected amounts of ED were: intracellular, 9.63 ± 1.6 pg per 500,000 cells; and extracellular, 10.25 ± 1.06 pg per 500,000 cells. Our findings show that human DPCs in vitro express immunoreactivity for ACTH, MSH, and ED. Among the detected POMC-derived peptides, however, only ED appears to be secreted by DPCs in detectable amounts in our hands.
Studies on ASIP expression in human DPCs in culture
Expression of AP has been demonstrated within the dermal papilla of agouti mice in which it antagonizes the effect of melanocortins on eumelanin synthesis in hair follicle melanocytes (38). To clarify whether human DPCs express the human homolog of AP, ASIP, we performed RT-PCR and Western immunoblot analysis using an antibody that detects both AP and ASIP (31). cDNA from human adult testis was used as a positive for RT-PCR analysis (38). For Western immunoblotting, recombinant AP (85% homologous with ASIP at the amino acid level) as well as crude protein lysate from human testis was used as a positive control (38). In human DPC extracts, neither ASIP transcripts nor protein was detectable (Fig. 2, A and B). In contrast, human testis cDNA generated a specific amplification product of the expected size of 385 bp (29) (Fig. 2A). Western immunoblotting furthermore revealed the presence of an immunoreactive band of approximately 21.5 kDa in protein extracts from human testis (Fig. 2B), suggesting that ASIP protein is constitutively expressed in human adult testis.
Precursor proteases in human DPCs in culture
The generation of POMC-derived peptides by human DPCs suggested the expression of members of the subtilisin/kexin family of PCs, which cleave POMC into POMC-derived peptides. These enzymes are PC1, PC2 (together with its essential cofactor 7B2), PACE4, and furin convertase (FC) (3). In contrast to the previously reported detection of PC1 and PC2 in human epidermal melanocytes and dermal fibroblasts (27, 39), only transcripts for PC2 and 7B2 but not for PC1 were detected in human DPCs in culture in our hands (Fig. 3, A, C, and D). The PC2- and 7B2-related amplification products in DPCs were of the expected size of 299 (PC2) and 454 bp (7B2), whereas contamination controls were negative. The absence of a PC1-related amplification product in human DPCs was apparently not due to technical reasons because cDNA from NHM used as positive control clearly generated the expected PC-1 amplicon (Fig. 3A). To further search for other members of the PC family that may process POMC into POMC-derived peptides in human DPCs, we analyzed the expression of PACE4 and FC. We could not detect RNA expression for PACE4 whose transcripts were readily detectable in epidermal keratinocytes used as a positive control (40) (Fig. 3B). In contrast, we detected the expression of FC at the RNA level as demonstrated by amplification of the expected 101-bp product whose identity with FC was confirmed by DNA sequencing (data not shown) (Fig. 3E). These data demonstrate that at least two members of the family of precursor proteases, PC2 plus its essential cofactor 7B2, and FC are expressed in human DPCs in culture.
Detection of MC-1R and MC-4R in human DPCs in vitro and in situ
To investigate whether human DPCs are target cells for melanocortins, we first determined the RNA expression profile of all known MC-R subtypes by RT-PCR. DPC in vitro expressed transcripts for both MC-1R and MC-4R, whereas transcripts for MC-2R, MC-3R, and MC-5R were absent (Fig. 4A). The MC-1R and MC-4R amplification products from DPCs comigrated with the amplicons retrieved from the positive controls, which were NHM for MC-1R (416 bp) and genomic fibroblast DNA for MC-4R (566 bp) as reported previously (25, 26). Contamination controls were negative (Fig. 4A). MC-1R and MC-4R expression by human DPCs was subsequently supported by immunofluorescence studies. Paraformaldehyde-fixed DPCs exhibited a specific punctate staining pattern for both MC-1R and MC-4R (Fig. 4B). No such staining was detectable in cells incubated with preimmune serum or an isotype control used at the same concentration as the primary antibody (Fig. 4B). To further determine whether the detected MC-1R and MC-4R immunoreactivity is located in the plasma membrane and/or the cytoplasm, we performed experiments with permeated vs. nonpermeated cells. To this end, DPCs were also stained with an antibody against SMA and DAPI, the latter a nuclear marker. SMA is a well-established marker for myofibroblast transdifferentiation and is thus used for identification of DPCs. In our case, the anti-SMA antibody was used as a cytoplasmic indicator that would be visible only in permeated cells, i.e. those with paraformaldehyde fixation and detergent treatment, but not in cells with surface fixation, i.e. fixation with paraformaldehyde only. The results of these experiments are shown in Fig. 4C. In nonpermeated cells triple staining for MC-1R, SMA, and DAPI generated only a discrete punctate red labeling (due to MC-1R immunoreactivity) and a diffuse greenish cytoplasmic stain (due to nonspecific binding of the anti-SMA antibody) (Fig. 4C). MC-1R immunostaining became accentuated in permeated cells in which SMA appeared as the characteristic filament network (Fig. 4C). Similarly, MC-4R immunoreactivity was more abundant in permeated than nonpermeated cells (Fig. 4C). The data indicate that immunoreactivity for both MC-1R and MC-4R in human DPCs is located in the plasma membrane as well as the cytoplasm, the latter likely to correspond to newly synthesized receptors being moved to the plasma membrane via endoplasmic reticulum, Golgi apparatus, and vesicular pathways.
To finally learn about the expression of MC-1R and MC-4R in DPCs in situ, we performed immunohistochemical studies on tissue sections of normal adult human scalp skin. MC-1R and MC-4R immunoreactivity was detectable within the cytoplasm of fibroblastic cells of the dermal papilla (Fig. 4D). No immunostaining was observed in negative controls in which the primary antibody was replaced by preimmune serum or the isotype control (Fig. 4D). These findings corroborate the previously detected MC-1R and MC-4R expression in human DPCs in culture and suggest that these cells are also targets for MSH in vivo.
Evidence for functionally active MC-Rs in human DPCs in vitro
We next assessed the biological relevance of MC-Rs expressed by human DPCs. We initially focused on the effect of MSH on intracellular cAMP because engagement of all types of MC-Rs with their cognate ligands results in immediate activation of adenylyl cyclase. Based on previous findings that showed a significant dose-dependent cAMP response of human dermal fibroblasts 20 min after MSH treatment (25), we also decided to use this time point for our studies with human DPCs. Multiple independent experiments (n > 6) with cells from different donors revealed that only MSH doses of 10–6 M elicited a robust increase in intracellular cAMP (Fig. 5A). As expected, the artificial cAMP inducer forskolin (1 μM) used as positive control also increased intracellular cAMP (Fig. 5A). Neither changes in the amount of FCS during the deprivation phase of the cells or preincubation with IBMX for 1 h nor modified time points of MSH treatment resulted in significant increases in intracellular cAMP at MSH concentrations beyond 10–6 M (data not shown). However, because previous studies had provided compelling evidence that the human MC-1R is activated in nanomolar amounts of MSH (5, 6), we wanted to further specify the MSH-mediated cAMP response in human DPCs. Therefore, cells were preincubated with 10–6 M of a synthetic peptide corresponding to the amino acids 87–132 of the carboxy-terminal domain of ASIP followed by stimulation with MSH at the same concentration. Previously it has been shown that cysteine-rich C-terminal ASIP fragments are as potent as full-length ASIP (41). As depicted in Fig. 5B, the MC-1R/MC-4R antagonistic peptide largely abrogated the MSH-mediated cAMP response in human DPCs. These findings strongly suggest that the observed cAMP response by MSH in human DPCs is mediated via activation of MC-1R and/or MC-4R, both of which are expressed in these cells as presented above.
To further assess the functionality of the MC-Rs expressed in human DPCs by yet another signal transduction pathway known to be activated by MSH, we investigated the changes in intracellular calcium, an event crucially involved in elicitation of the biological effect of MSH (42). Recently rapid and acute increase in intracellular calcium has also been described in normal human keratinocytes and HaCaT cells after stimulation with MSH and ACTH peptides (43). In accordance with the detection of calcium spikes induced by MSH in keratinocytes, treatment of human DPCs with MSH resulted in a significant intracellular calcium spike within 10 sec. Both MSH doses of 10–6 M (data not shown) but also 10–8 M consistently evoked this response, whereas MSH at 10–10 M did not have any effect (Fig. 5C). The calcium spikes induced by MSH were transient and returned to baseline levels by 60 sec. As expected, treatment of the cells with inomycin (10–4 M) used as positive control also led to a robust calcium spike in human DPCs (Fig. 5C). These findings show that in addition to the cAMP pathway, the calcium signaling pathway is activated by MSH in human DPCs.
Because MSH has been reported to exhibit immunoregulatory activities (44, 45, 46) and aberrant expression of ICAM-1 has been implicated in the pathogenesis of alopecia areata (47, 48, 49), an inflammatory, nondestructive type of hair loss, we finally investigated whether MSH can modulate basal and cytokine-induced ICAM-1 expression in human DPCs in vitro. IFN, a prototypical inducer of ICAM-1 (50), strongly induced mRNA expression of ICAM-1 as shown by RT-PCR (Fig. 5D). MSH alone did not have any modulatory activity on basal expression of ICAM-1. When coincubated with IFN, MSH at 10–6 M significantly suppressed the inductive effect of IFN on ICAM-1 expression. In accordance with our data on the cAMP induction by MSH in human DPCs, concentrations of 10–8 M failed to suppress the inductive effect of IFN on ICAM-1 (data not shown).
Collectively, these findings indicate that the MC-Rs expressed by human DPCs in vitro are functionally active. MSH triggers at least two classical signal transduction pathways in these cells, the cAMP and calcium pathway. In addition, MSH exerts immunoregulatory effects in these cells such as suppression of adhesion molecule expression induced by a prototypical proinflammatory cytokine.
Discussion
In this study we report the detection of several components of the POMC system in human DPCs, i.e. POMC, transcripts for distinct members of the PC family of precursor proteases, immunoreactivity for POMC-derived peptides, and the presence of receptors for MSH.
Most importantly, the receptors for MSH, MC-1R, and MC-4R appear to be functionally active in human DPCs because MSH elicits an increase in intracellular cAMP and calcium as well as down-regulates the expression of ICAM-1 induced by IFN. Concomitant expression of different MC-Rs has been described in several cutaneous cell types. Human epidermal melanocytes were shown to express not only MC-1R (51) but also MC-2R at the RNA level (52). Likewise, expression of MC-1R and MC-2R was reported in human keratinocytes (53, 54). These findings are supported by the detection of MC-2R at the RNA level in whole human and murine skin (10, 52). In addition, dual expression of MC-2R and MC-5R has been reported in the murine 3T3-L1 adipocyte cell line (55). We are unaware of any report showing expression of MC-4R in human skin. Expression of MC-4R is primarily found in the brain. However, MC-4R has recently been reported to occur in a number of extraneural tissues including the skin in the fetal rat, suggesting a role of MC-4R beyond appetite, weight control, and regulation of linear growth (56). The biological function of dual expression of different MC-R subtypes is enigmatic. The human MC-1R has the highest affinity for MSH and an almost equal affinity for ACTH, whereas the MC-4R is equipotently activated by both melanocortins (6). It is possible that the relative level of MC-Rs expressed dictates the biological response of a cell to the ligand due to MC-R signal transduction heterogeneity. It has been reported that albeit both MC-3R and MC-4R activate adenylyl cyclase, activation of the MAPK cascade is induced only by MC-4R (57). Because the used ASIP peptide in our studies was antagonistic to both MC-1R and MC-4R, further research is needed to dissect the signaling pathways attributable to each MC-R subtype expressed by human DPCs. Future studies will also have to clarify why the cAMP response (as well as the immunoregulatory effect) of MSH is induced only at 10–6 M. The melanotropic response of MSH in normal human melanocytes is typically induced at nanomolar doses as predicted by the MC-1R binding affinity (51). Ligand binding studies should help to determine the actual number of MC-Rs being expressed on the surface of human DPCs.
Regarding the immunoregulatory effect of MSH on the expression of ICAM-1 in human DPCs, our findings extend those of others who also reported regulatory activities of MSH on the expression of adhesion molecules. Accordingly, MSH was shown to suppress ICAM-1 expression induced by TNF in normal and transformed human melanocytes (58). MSH also suppressed lipopolysaccharide-induced expression of ICAM-1, vascular adhesion molecule-1, and E-selectin by dermal endothelial cells in a mouse model of experimentally induced vasculitis (59). Ito et al. (60) recently demonstrated that MSH can suppress ectopically induced MHC class I and II expression in organ-cultured anagen human hair bulbs. Typically the dermatohistopathologic picture of the hair follicles in patients with alopecia areata consists of peribulbar T lymphocytes forming a bee swarm-like infiltrate. In light of the collapse of the immune privilege in alopecia areata leading to aberrant adhesion molecule expression (61) and the presence of functional MC-Rs in DPCs, it will be worthwhile to examine the expression and functionality of MC-Rs in DPCs of patients with this disorder. In addition, it will be interesting to examine the expression of POMC, POMC-derived peptides, and PCs in this condition as already suggested by others (62).
Unexpectedly we have been unable to detect immunoreactive amounts of ACTH and MSH by means of immunometric assays in human DPCs in culture, although these cells expressed immunoreactivity for both of these POMC peptides as shown by immunofluorescence studies. This is in contrast to epidermal melanocytes and keratinocytes (36), human dermal fibroblasts (27), and microvascular endothelial cells (63), all of which release immunoreactive amounts of melanocortins into the culture media. Biochemical studies using reversed-phase HPLC as well as liquid chromatography-mass spectrometry confirmed the presence of the above POMC peptides in cultured human melanocytes, melanoma, and squamous cell carcinoma cells as well as normal and diseased human skin (64, 65, 66). At present, we cannot exclude that MSH and ACTH produced by human DPCs are rapidly degraded in vitro and thus escape from detection. Interestingly, the ACTH immunoreactivity observed in human DPCs appeared to be nuclear and could be generated by two antibodies directed against two different epitopes of ACTH. Because preincubation with ACTH completely abrogated the immunostaining, it is most likely that the used antibodies detect ACTH or closely related ACTH peptides. Of note, nuclear localization has been reported for a number of growth factors and hormones including pro-CRH (67). However, MSH immunoreactivity in DPCs was found within the cytoplasm, which is puzzling in light of the nuclear staining of ACTH and the known processing of POMC. Therefore, additional studies such as immunofluorescence analysis with different fixation methods and use of organelle markers, subcellular fractionation with biochemical analysis of ACTH, and immune electron microscopy should define the exact localization of ACTH in human DPC. In contrast to MSH and ACTH, we were able to detect ED immunoreactivity and immunoreactive amounts of this POMC peptide by radioimmune assay in the culture media of ACTH and immune human DPCs. It needs to be determined whether human DPCs indeed secrete bioactive amounts of this neuropeptide.
Answering this question is necessary because ED is posttranslationally modified by N-acetylation to yield inactive forms of the peptide (68, 69). Given the detected expression of PC2, 7B2, and FC in human DPCs and the enzymatic activity of these enzymes against POMC (3), it is conceivable that these enzymes may be involved in generation of ED. However, during revision of this manuscript, Kauser et al. (70) reported on the expression of the POMC system in the human hair follicle. These authors detected the expression of both PC1 and PC2 in cultured DPCs and demonstrated immunoreactivity for both enzymes in the human dermal papilla in situ. In murine skin, immunoreactivity for PC1 and PC2 was present in epidermal keratinocytes and the sebaceous unit as well as some adjacent cells (13). Both PC1 and PC2 displayed a differential temporal and spatial pattern of immunoreactivity. The reason for the discrepancy between our findings and those of others (70) is unclear but may be related to donor variability and/or differences in the cell culture conditions.
In summary, we have presented herein evidence for functionally active receptors for MSH in human DPCs. In addition, we have detected several components of the POMC system in these cells. Our data as well as those from others (70) suggest close interconnections via the POMC system among the cellular components of the hair follicle as depicted in Fig. 6. Via MC-1R and MC-4R, MSH (and possibly other melanocortins) may regulate inflammatory reactions such as expression of ICAM-1 in DPCs, thereby modulating the recruitment of T lymphocytes into the dermal papilla. On the other hand, POMC-derived peptides presumably released by DPCs may act as paracrine regulators of hair follicle epithelia and melanocytes. For example, MSH, ACTH, and ED released by DPCs may stimulate melanogenesis because human hair follicle melanocytes express not only MC-1R but also the μ-opioid receptor (70, 71). Because interfollicular keratinocytes express μ-opioid receptor (72) as well and respond to ED with increased cytokeratin 16 expression (73), it is possible that ED released from DPCs may regulate keratinization of the hair follicle in analogy. In light of the diversity of biological actions of MSH such as regulation of extracellular matrix composition and apoptosis (25, 74), future studies can be expected to add novel insight into our understanding of the POMC system and the hair follicle.
Acknowledgments
The authors acknowledge the expert technical assistance of Ilka Wolff.
Footnotes
This work was supported by a research grant from the Deutsche Forschungsgemeinschaft (DFG, 1075/5-1, to M.B.) and a research grant on alopecia from Pfizer.
Abbreviations: AP, Agouti protein; ASIP, agouti signaling protein; DAPI, 4',6-diamido-2-phenylindole dihydrochloride; DPC, dermal papilla cell; ED, endorphin; FC, furin convertase; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBRS, HEPES-buffered Ringer solution; IBMX, isobutyl methylxanthine; ICAM, intercellular adhesion molecule; IFN, interferon; MC-R, melanocortin receptor; NHM, normal human melanocyte; PACE4, paired basic amino acid cleaving enzyme 4 gene; PC, prohormone convertase; POMC, proopiomelanocortin; SMA, -smooth muscle actin.
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