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• W2007200965 abstract "In the last few years it has become apparent that the skin is a locoregional source for several proopiomelanocortin-derived peptides including α-melanocyte-stimulating hormone, adrenocorticotropin, and β-endorphin. The enzymes that regulate expression of these neuropeptides are the prohormone convertases 1 and 2. In this study we demonstrate, by reverse transcriptase polymerase chain reaction and Western immunoblotting, that cultured human dermal fibroblasts express prohormone convertases 1 and 2 as well as 7B2, which is an essential cofactor for enzymatic activity of prohormone convertase 2. Immunofluorescence studies revealed prohormone convertase 1 to be mainly expressed in the perinuclear region in vesicular structures resembling the trans-Golgi network, whereas prohormone convertase 2 was found in the trans-Golgi network as well as in vesicular structures diffusely distributed in the peripheral cytoplasm. Expression of both enzymes was also confirmed in fibroblasts of normal adult human skin by immunohistochemistry using antibodies against prohormone convertases 1 and 2 and vimentin. To assess the relevance of prohormone convertase 1 and 2 expression in human dermal fibroblasts, we studied the expression of proopiomelanocortin and proopiomelanocortin-derived peptides. Proopio melanocortin expression was detected by reverse transcriptase polymerase chain reaction and Western immunoblotting. α-Melanocyte-stimulating hormone, adrenocorticotropin, and β-endorphin were mainly located in vesicular structures as demonstrated by immunofluorescence. Production of these peptides was confirmed by radioimmunoassay, immunoradiometric assay, or enzyme immunoassay. Among several stimuli tested, interleukin-1 was found to upregulate production of α-melanocyte-stimulating hormone in human dermal fibroblasts. In summary, we have shown that human dermal fibroblasts express the enzymatic machinery for proopiomelanocortin processing and make proopiomelanocortin, α-melanocyte-stimulating hormone, adrenocorticotropin, and β-endorphin. Production of proopiomelanocortin peptides by human dermal fibroblasts may be relevant for fibroblast functions such as collagen degradation and/or regulation of dermal immune responses. In the last few years it has become apparent that the skin is a locoregional source for several proopiomelanocortin-derived peptides including α-melanocyte-stimulating hormone, adrenocorticotropin, and β-endorphin. The enzymes that regulate expression of these neuropeptides are the prohormone convertases 1 and 2. In this study we demonstrate, by reverse transcriptase polymerase chain reaction and Western immunoblotting, that cultured human dermal fibroblasts express prohormone convertases 1 and 2 as well as 7B2, which is an essential cofactor for enzymatic activity of prohormone convertase 2. Immunofluorescence studies revealed prohormone convertase 1 to be mainly expressed in the perinuclear region in vesicular structures resembling the trans-Golgi network, whereas prohormone convertase 2 was found in the trans-Golgi network as well as in vesicular structures diffusely distributed in the peripheral cytoplasm. Expression of both enzymes was also confirmed in fibroblasts of normal adult human skin by immunohistochemistry using antibodies against prohormone convertases 1 and 2 and vimentin. To assess the relevance of prohormone convertase 1 and 2 expression in human dermal fibroblasts, we studied the expression of proopiomelanocortin and proopiomelanocortin-derived peptides. Proopio melanocortin expression was detected by reverse transcriptase polymerase chain reaction and Western immunoblotting. α-Melanocyte-stimulating hormone, adrenocorticotropin, and β-endorphin were mainly located in vesicular structures as demonstrated by immunofluorescence. Production of these peptides was confirmed by radioimmunoassay, immunoradiometric assay, or enzyme immunoassay. Among several stimuli tested, interleukin-1 was found to upregulate production of α-melanocyte-stimulating hormone in human dermal fibroblasts. In summary, we have shown that human dermal fibroblasts express the enzymatic machinery for proopiomelanocortin processing and make proopiomelanocortin, α-melanocyte-stimulating hormone, adrenocorticotropin, and β-endorphin. Production of proopiomelanocortin peptides by human dermal fibroblasts may be relevant for fibroblast functions such as collagen degradation and/or regulation of dermal immune responses. β-endorphin human dermal fibroblasts melanocyte-stimulating hormone normal human melanocytes prohormone convertase proopiomelanocortin trans-Golgi network wheat germ agglutinin Proopiomelanocortin (POMC) peptides comprise a number of bioactive substances that are derived from a large precursor prohormone of approximately 31 kDa. Two prohormone convertases, PC1 and PC2, belonging to an evolutionary conserved family of serine proteinases of the subtilisin/kexin type, have been found to be associated with POMC peptide expression (Seidah et al., 1999Seidah N.G. Benjannet S. Hamelin J. et al.The subtilisin/kexin family of precursor convertases. Emphasis on PC1, PC2/7B2, POMC and the novel enzyme SKI-1.Ann N Y Acad Sci. 1999; 885: 57-74Crossref PubMed Scopus (118) Google Scholar). Whereas PC1 activity results in adrenocorticotropin (ACTH) and β-lipotropin formation, PC2 catalyzes the formation of α-melanocyte-stimulating hormone (α-MSH), β-endorphin (β-ED), and corticotrophin-like intermediate lobe peptide (Benjannet et al., 1991Benjannet S. Rondeau N. Day R. Chretien M. Seidah N.G. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues.Proc Natl Acad Sci USA. 1991; 88: 3564-3568Crossref PubMed Scopus (521) Google Scholar;Seidah et al., 1999Seidah N.G. Benjannet S. Hamelin J. et al.The subtilisin/kexin family of precursor convertases. Emphasis on PC1, PC2/7B2, POMC and the novel enzyme SKI-1.Ann N Y Acad Sci. 1999; 885: 57-74Crossref PubMed Scopus (118) Google Scholar). PC2 activity itself is partly regulated by a chaperone-like binding protein named 7B2, which facilitates zymogene activation of the precursor PC2 (Benjannet et al., 1998Benjannet S. Mamarbachi A.M. Hamelin J. Savaria D. Munzer J.S. Chretien M. Seidah N.G. Residues unique to the pro-hormone convertase PC2 modulate its autoactivation, binding to 7B2 and enzymatic activity.FEBS Lett. 1998; 428: 37-42Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar;Apletalina et al., 2000Apletalina E.V. Muller L. Lindberg I. Mutations in the catalytic domain of prohormone convertase 2 result in decreased binding to 7B2 and loss of inhibition with 7B2 C-terminal peptide.J Biol Chem. 2000; 275: 14667-14677Crossref PubMed Scopus (14) Google Scholar). 7B2 is also required for development of the full enzymatic activity of PC2 (Zhu and Lindberg, 1995Zhu X. Lindberg I. 7B2 facilitates the maturation of proPC2 in neuroendocrine cells and is required for the expression of enzymatic activity.J Cell Biol. 1995; 129: 1641-1650Crossref PubMed Scopus (142) Google Scholar). POMC peptides elicit a plethora of biologic effects including behavioral, cognitive, immunomodulating, antimicrobial, pigmentary, nutritional, and thermoregulatory effects (Hadley and Haskell-Luevano, 1999Hadley M.E. Haskell-Luevano C. The proopiomelanocortin system.Ann N Y Acad Sci. 1999; 885: 1-21Crossref PubMed Scopus (123) Google Scholar;Luger et al., 1999bLuger T.A. Paus R. Slominski A. Lipton J. The proopiomelanocortin system in cutaneous neuroimmunomodulation. An introductory overview.Ann N Y Acad Sci. 1999; 885: xi-xivCrossref PubMed Scopus (33) Google Scholar;Cutuli et al., 2000Cutuli M. Cristiani S. Lipton J.M. Catania A. Antimicrobial effects of α-MSH peptides.J Leukoc Biol. 2000; 67: 233-239PubMed Google Scholar). With regard to the skin, α-MSH has attained most attraction as it has originally been characterized as an important regulator of the coat color in many vertebrate species (Lerner and McGuire, 1961Lerner A.B. McGuire J.S. Effect of α- and β-melanocyte-stimulating hormones on skin color of man.Nature. 1961; 189: 176-179Crossref PubMed Scopus (228) Google Scholar;Thody and Graham, 1998Thody A.J. Graham A. Does α-MSH have a role in regulating skin pigmentation in humans?.Pigment Cell Res. 1998; 11: 265-274Crossref PubMed Scopus (92) Google Scholar). In addition, an increasing number of studies demonstrated that α-MSH exerts potent immunoregulatory effects, which may be relevant for cutaneous immune responses and inflammation (Slominski et al., 2000Slominski A. Wortsman J. Luger T. Paus R. Solomon S. Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress.Physiol Rev. 2000; 80: 979-1020Crossref PubMed Scopus (597) Google Scholar). α-MSH suppresses the production of certain immunoregulatory and proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, interferon-γ, and tumor necrosis factor α (TNF-α) (Catania and Lipton, 1993Catania A. Lipton J.M. α-Melanocyte-stimulating hormone in the modulation of host reactions.Endocr Rev. 1993; 14: 564-576PubMed Google Scholar;Luger et al., 1993Luger T.A. Schauer E. Trautinger F. Krutmann J. Ansel J. Schwarz A. Schwarz T. Production of immunosuppressing melanotropins by human keratinocytes.Ann N Y Acad Sci. 1993; 680: 567-570Crossref PubMed Scopus (57) Google Scholar;Luger et al., 1999aLuger T.A. Kalden D. Scholzen T.E. Brzoska T. α-Melanocyte-stimulating hormone as a mediator of tolerance induction.Pathobiology. 1999; 67: 318-321Crossref PubMed Scopus (35) Google Scholar). Expression of the costimulatory molecules CD86 and CD40 is suppressed by α-MSH (Bhardwaj et al., 1997Bhardwaj R. Becher E. Mahnke K. Hartmeyer M. Schwarz T. Scholzen T. Luger T.A. Evidence for the differential expression of the functional α-melanocyte-stimulating hormone receptor MC-1 on human monocytes.J Immunol. 1997; 158: 3378-3384PubMed Google Scholar). α-MSH induces the production of the cytokine synthesis inhibitory factor IL-10 in human monocytes (Bhardwaj et al., 1996Bhardwaj R.S. Schwarz A. Becher E. Mahnke K. Aragane Y. Schwarz T. Luger T.A. Proopiomelanocortin-derived peptides induce IL-10 production in human monocytes.J Immunol. 1996; 156: 2517-2521PubMed Google Scholar;Becher et al., 1999Becher E. Mahnke K. Brzoska T. Kalden D.H. Grabbe S. Luger T.A. Human peripheral blood-derived dendritic cells express functional melanocortin receptor MC-1R.Ann N Y Acad Sci. 1999; 885: 188-195Crossref PubMed Scopus (58) Google Scholar). Depending on the cell type studied, α-MSH can downregulate lipopolysaccharide- or TNF-α-induced expression of the vascular cellular adhesion molecule 1 and intercellular adhesion molecule 1 (Hedley et al., 1998Hedley S.J. Gawkrodger D.J. Weetman A.P. Morandini R. Boeynaems J.M. Ghanem G. Neil S.M. α-Melanocyte-stimulating hormone inhibits TNF-α stimulated intercellular adhesion molecule-1 expression in normal cutaneous human melanocytes and in melanoma cell lines.Br J Dermatol. 1998; 138: 536-543Crossref PubMed Scopus (41) Google Scholar;Morandini et al., 1998Morandini R. Boeynaems J.M. Hedley S.J. MacNeil S. Ghanem G. Modulation of ICAM-1 expression by α-MSH in human melanoma cells and melanocytes.J Cell Physiol. 1998; 175: 276-282https://doi.org/10.1002/(sici)1097-4652(199806)175:3<276::aid-jcp5>3.3.co;2-5Crossref PubMed Scopus (0) Google Scholar;Kalden et al., 1999Kalden D.H. Scholzen T. Brzoska T. Luger T.A. Mechanisms of the antiinflammatory effects of α-MSH. Role of transcription factor NF-κB and adhesion molecule expression.Ann N Y Acad Sci. 1999; 885: 254-261Crossref PubMed Scopus (77) Google Scholar) as well as lipopolysaccharide-mediated activation of the transcription factor NF-κB (Manna and Aggarwal, 1998Manna S.K. Aggarwal B.B. α-Melanocyte-stimulating hormone inhibits the nuclear transcription factor NF-κB activation induced by various inflammatory agents.J Immunol. 1998; 161: 2873-2880PubMed Google Scholar;Brzoska et al., 1999Brzoska T. Kalden D.H. Scholzen T. Luger T.A. Molecular basis of the α-MSH/IL-1 antagonism.Ann N Y Acad Sci. 1999; 885: 230-238Crossref PubMed Scopus (50) Google Scholar;Kalden et al., 1999Kalden D.H. Scholzen T. Brzoska T. Luger T.A. Mechanisms of the antiinflammatory effects of α-MSH. Role of transcription factor NF-κB and adhesion molecule expression.Ann N Y Acad Sci. 1999; 885: 254-261Crossref PubMed Scopus (77) Google Scholar). These effects may represent parts of the molecular mechanisms by which α-MSH suppresses the induction as well as the elicitation phase in a mouse model of contact hypersensitivity, or by which α-MSH suppresses the experimentally provoked cutaneous vasculitis induced by lipopolysaccharide (Grabbe et al., 1996Grabbe S. Bhardwaj R.S. Mahnke K. Simon M.M. Schwarz T. Luger T.A. α-Melanocyte-stimulating hormone induces hapten-specific tolerance in mice.J Immunol. 1996; 156: 473-478PubMed Google Scholar;Sunderkötter et al., 1999Sunderkötter C. Kalden H. Brzoska T. Sorg C. Luger T.A. α-MSH reduces vasculitis in the local Shwartzman reaction.Ann N Y Acad Sci. 1999; 885: 414-418Crossref PubMed Scopus (9) Google Scholar). In contrast to α-MSH, the biologic activities of ACTH and β-ED in the skin are less defined. ACTH, beyond its ability to induce pigmentation (Wintzen and Gilchrest, 1996Wintzen M. Gilchrest B.A. Proopiomelanocortin, its derived peptides, and the skin.J Invest Dermatol. 1996; 106: 3-10Crossref PubMed Scopus (123) Google Scholar;Thody and Graham, 1998Thody A.J. Graham A. Does α-MSH have a role in regulating skin pigmentation in humans?.Pigment Cell Res. 1998; 11: 265-274Crossref PubMed Scopus (92) Google Scholar;Hirobe and Abe, 2000Hirobe T. Abe H. ACTH4-12 is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in serum-free primary culture.J Exp Zool. 2000; 286: 632-640https://doi.org/10.1002/(sici)1097-010x(20000501)286:6<632::aid-jez10>3.0.co;2-4Crossref PubMed Google Scholar), can regulate lipogenesis in murine adipocytes as the receptor for ACTH has been identified on these cells (Boston and Cone, 1996Boston B.A. Cone R.D. Characterization of melanocortin receptor subtype expression in murine adipose tissues and in the 3T3-L1 cell line.Endocrinology. 1996; 137: 2043-2050Crossref PubMed Scopus (174) Google Scholar;Boston, 1999Boston B.A. The role of melanocortins in adipocyte function.Ann N Y Acad Sci. 1999; 885: 75-84Crossref PubMed Scopus (76) Google Scholar). Increased plasma levels of β-ED were found in patients with psoriasis, atopic dermatitis, and systemic sclerosis, and in individuals exposed to ultraviolet (UV) light (Levins et al., 1983Levins P.C. Carr D.B. Fisher J.E. Momtaz K. Parrish J.A. Plasma β-endorphin and β-lipoprotein response to ultraviolet radiation.Lancet. 1983; 2: 166Abstract PubMed Scopus (60) Google Scholar;Glinski et al., 1994Glinski W. Brodecka H. Glinska-Ferenz M. Kowalski D. Increased concentration of β-endorphin in sera of patients with psoriasis and other inflammatory dermatoses.Br J Dermatol. 1994; 131: 260-264Crossref PubMed Scopus (34) Google Scholar). In situ expression of β-ED on the protein level was pronounced in dysplastic nevus, basal cell carcinoma, keratoacanthoma, and psoriatic skin as demonstrated by immunostaining (Slominski et al., 1993Slominski A. Wortsman J. Mazurkiewicz J.E. et al.Detection of proopiomelanocortin-derived antigens in normal and pathologic human skin.J Lab Clin Med. 1993; 122: 658-666PubMed Google Scholar). Detection of µ-opioid receptors in a variety of cutaneous cell types further suggests an important role of β-ED in skin physiology and a bidirectional neuroimmuno-dermatologic axis (Bigliardi et al., 1998Bigliardi P.L. Bigliardi-Qi M. Buechner S. Rufli T. Expression of µ-opiate receptor in human epidermis and keratinocytes.J Invest Dermatol. 1998; 111: 297-301https://doi.org/10.1046/j.1523-1747.1998.00259.xCrossref PubMed Scopus (93) Google Scholar;Bigliardi-Qi et al., 2000Bigliardi-Qi M. Bigliardi P.L. Eberle A.N. Buchner S. Rufli T. β-Endorphin stimulates cytokeratin 16 expression and downregulates µ-opiate receptor expression in human epidermis.J Invest Dermatol. 2000; 114: 527-532https://doi.org/10.1046/j.1523-1747.2000.00801.xCrossref PubMed Scopus (57) Google Scholar). It is established that POMC peptides are widely expressed in several extraneural tissues including the skin (Thody et al., 1983Thody A.J. Ridley K. Penny R.J. Chalmers R. Fisher C. Shuster S. MSH peptides are present in mammalian skin.Peptides. 1983; 4: 813-816Crossref PubMed Scopus (158) Google Scholar;Blalock, 1999Blalock J.E. Proopiomelanocortin and the immune-neuroendocrine connection.Ann N Y Acad Sci. 1999; 885: 161-172Crossref PubMed Scopus (76) Google Scholar;Solomon, 1999Solomon S. POMC-derived peptides and their biological action.Ann N Y Acad Sci. 1999; 885: 22-40Crossref PubMed Scopus (56) Google Scholar). POMC mRNA and various POMC peptides are present in normal and human diseased skin (Slominski et al., 1993Slominski A. Wortsman J. Mazurkiewicz J.E. et al.Detection of proopiomelanocortin-derived antigens in normal and pathologic human skin.J Lab Clin Med. 1993; 122: 658-666PubMed Google Scholar;Nagahama et al., 1998Nagahama M. Funasaka Y. Fernandez-Frez M.L. Ohashi A. Chakraborty A.K. Ueda M. Ichihashi M. Immunoreactivity of α-melanocyte-stimulating hormone, adrenocorticotrophic hormone and β-endorphin in cutaneous malignant melanoma and benign melanocytic naevi.Br J Dermatol. 1998; 138: 981-985Crossref PubMed Scopus (50) Google Scholar), and several skin cell types produce α-MSH and ACTH (Schauer et al., 1994Schauer E. Trautinger F. Kock A. et al.Proopiomelanocortin-derived peptides are synthesized and released by human keratinocytes.J Clin Invest. 1994; 93: 2258-2262Crossref PubMed Scopus (309) Google Scholar;Slominski et al., 1996Slominski A. Ermak G. Hwang J. Mazurkiewicz J. Corliss D. Eastman A. The expression of proopiomelanocortin (POMC) and of corticotropin releasing hormone receptor (CRH-R) genes in mouse skin.Biochim Biophys Acta. 1996; 1289: 247-251Crossref PubMed Scopus (80) Google Scholar;Wintzen and Gilchrest, 1996Wintzen M. Gilchrest B.A. Proopiomelanocortin, its derived peptides, and the skin.J Invest Dermatol. 1996; 106: 3-10Crossref PubMed Scopus (123) Google Scholar) and β-ED (Levins et al., 1983Levins P.C. Carr D.B. Fisher J.E. Momtaz K. Parrish J.A. Plasma β-endorphin and β-lipoprotein response to ultraviolet radiation.Lancet. 1983; 2: 166Abstract PubMed Scopus (60) Google Scholar;Zanello et al., 1999Zanello S.B. Jackson D.M. Holick M.F. An immunocytochemical approach to the study of β-endorphin production in human keratinocytes using confocal microscopy.Ann N Y Acad Sci. 1999; 885: 85-99Crossref PubMed Scopus (30) Google Scholar). With regard to dermal fibroblasts,Teofoli et al., 1997Teofoli P. Motoki K. Lotti T.M. Uitto J. Mauviel A. Propiomelanocortin (POMC) gene expression by normal skin and keloid fibroblasts in culture: modulation by cytokines.Exp Dermatol. 1997; 6: 111-115Crossref PubMed Scopus (37) Google Scholarhave recently detected POMC expression on the mRNA level; however, nothing is known about the enzyme machinery that generates the mature POMC products α-MSH, ACTH, and β-ED (Teofoli et al., 1999Teofoli P. Frezzolini A. Puddu P. De Pita O. Mauviel A. Lotti T. The role of proopiomelanocortin-derived peptides in skin fibroblast and mast cell functions.Ann N Y Acad Sci. 1999; 885: 268-276Crossref PubMed Scopus (30) Google Scholar). In this paper we demonstrate that human dermal fibroblasts (HDF) in culture and in situ express PC1 and PC2. Moreover, we have examined POMC expression as well as several stimuli that may regulate POMC peptide production and secretion. HDF derived from neonatal foreskin were purchased from BioWhittaker (Walkersville, MD) and maintained in RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS), 1% glutamine, and 1% penicillin/streptomycin (all from Biochrom) in a humidified atmosphere of 5% CO2. The cells were used in passages 3–8 only. Normal human melanocytes (NHM) were purchased from BioWhittaker and cultured with all supplements according to the manufacturer. Total RNA was isolated from HDF and NHM using an SV Total RNA isolation kit from Promega, Madison, WI. To avoid DNA contamination the extracted RNA was additionally treated with 10 U RNAse-free DNase I for 1 h at 37°C (Promega). For RT-PCR, 2 µg of total RNA was reverse transcribed using oligo(dT) primers and AMV Reverse Transcriptase (Promega). PCR amplification was performed with REDTaq polymerase (Sigma, Taufkirchen, Germany) and commercially synthesized specific primer pairs (Gibco-BRL, Gaithersburg, MD) as listed below. The reaction mixture contained 1 × REDTaq PCR buffer (10 × 100 mM Tris-HCl, pH 8.3, 500 mM KCl, 11 mM MgCl2, and 0.1% gelatin), 0.05 unit per µl REDTaq DNA polymerase (both Sigma), 0.2 mM dNTP (each) mixture (Promega), and 40 pmol of each primer. Nucleotide sequences for PCR primers and amplification programs were as follows: PC1 was amplified with the sense primer 5′-AGCAAACCCAAATCTCACCTG-3′ and the antisense primer 5′-TCTCCACCCCTCTTCTGTCAT-3′ yielding a 674 bp cDNA by one cycle at 94°C for 10 min, 53°C for 45 s, 72°C for 1 min, followed by 33 cycles at 94°C for 45 s, 53°C for 45 s, 72°C for 1 min, and a final cycle at 94°C for 45 s, 53°C for 45 s, 72°C for 10 min; PC2 was amplified with the sense primer 5′-AACGCA ACCAGAAGAGGAGA-3′ and the antisense primer 5′-ATGGCCAAC TTGGACTGGTA-3′ yielding a 299 bp cDNA by one cycle at 94°C for 5 min, followed by 38 cycles at 94°C for 30 s, 68°C for 45 s, 72°C for 45 s, and a final cycle at 94°C for 30 s, 68°C for 45 s, 72°C for 10 min; 7B2 was amplified using the sense primer 5′-CACCAGGCCATG AATCTT-3′ and the antisense primer 5′-CTGGATCCTTATCCT CATCTG-3′ yielding a 454 bp cDNA by one cycle at 94°C for 10 min, 68°C for 1 min, 72°C for 1 min, followed by 33 cycles at 94°C for 45 s, 68°C for 45 s, 72°C for 1 min, and a final cycle at 94°C for 45 s, 68°C for 45 s, 72°C for 10 min; POMC was amplified by the primer pairs previously described by Slominski and a modified protocol with one cycle at 94°C for 10 min, 68°C for 45 s min, 72°C for 1 min, 33 cycles at 94°C for 45 s, 68°C for 45 s, 72°C for 1 min, and a final cycle at 94°C for 45 s, 68°C for 45 s, 72°C for 10 min (Slominski et al., 1995Slominski A. Ermak G. Hwang J. Chakraborty A. Mazurkiewicz J.E. Mihm M. Proopiomelanocortin corticotropin releasing hormone and corticotropin releasing hormone receptor genes are expressed in human skin.FEBS Lett. 1995; 374: 113-116Abstract Full Text PDF PubMed Scopus (151) Google Scholar). To exclude that RNA samples were contaminated with genomic DNA, RNA samples that were not reverse transcribed as well as samples without cDNA were amplified. PCR products were separated electrophoretically on 1.5% TAE-agarose gels, stained with ethidium bromide, and photographed under UV. HDF and NHM were scraped into lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM ethyleneglycol-bis(β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid, 100 mM NaF, 0.01% NaN2) supplemented with protease inhibitors (10 µg per ml aprotinin, 5 µg per ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) as described previously (Böhm et al., 1995Böhm M. Moellmann G. Cheng E. Alvarez-Franco M. Wagner S. Sassone-Corsi P. Halaban R. Identification of p90RSK as the probable CREB-Ser133 kinase in human melanocytes.Cell Growth Differ. 1995; 6: 291-302PubMed Google Scholar). Protein concentration was measured by the modified Bradford assay (Bio-Rad, Richmond, CA). Whole cell lysates from SK-N-MC cells were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Identical amounts of lysate proteins (usually 30 µg per lane) were separated by 12% or 4%-12% gradient NuPAGE (Invitrogen, Carlsbad, CA) and electroblotted onto polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked overnight with 10% bovine serum albumin (BSA) and immunoprobed at 4°C overnight with anti-PC1 (Chemicon, Temecula, CA), anti-PC2 (1:500), or anti-7B2 (1:1000). The last two antibodies were generous gifts from Dr. N. Seidah, Montreal, Canada. POMC was detected using an anti-ACTH antibody (Sigma) directed against a common epitope of both ACTH and POMC (ACTH18-39). Membranes were incubated for 20 min with a horseradish-peroxidase-conjugated donkey anti-rabbit IgG antibody (Amersham Life Science, Freiburg, Germany). Antibody-antigen complexes were visualized by the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Cells seeded into eight-well tissue chambers (Laboratory-Tek, Nalge Nunc Int, Naperville, IL) were fixed and permeated with methanol for 30 min at -20°C. Unspecific binding was blocked with 5% donkey serum for 1 h at room temperature. Cells were then incubated for 2 h at room temperature with anti-PC1 (1:100) and anti-PC2 (1:200), both from Alexis, San Diego, CA; anti-α-MSH (1:50, Peninsula, San Carlos, CA) with no crossreactivity against β-MSH, γ-MSH, ACTH, Met-enkephalin, α-, β-, or γ-ED; anti-ACTH (1:100, Sigma) reactive against ACTH 18–39 and ACTH 1–39, but not against ACTH 1–17 and ACTH 1–10; and anti-β-ED (1:100, DRG, Marburg, Germany) with no crossreactivity against α-MSH, ACTH, or Met-enkephalin. After washing, bound antibodies were visualized by secondary donkey anti-rabbit antibodies conjugated with Texas Red (1:100, Dianova, Hamburg, Germany). In some experiments, the secondary antibody was coincubated with fluorescein isothiocyanate (FITC) conjugated wheat germ agglutinin (WGA) 1:250, Sigma. After three final washes, slides were mounted in Mowiol (Hoechst, Frankfurt, Germany) and stored at -20°C until use. For negative controls, whole rabbit serum was used instead of the primary antibody or the primary antibody was omitted. Cells were imaged with a confocal laser scanning microscope (TCS E, Leica, Heidelberg, Germany). Red and green fluorescence were excited with the 568 nm and 488 nm lines of an air-cooled Ar-Kr laser, and emissions were measured beyond 590 nm and 510 nm. Data acquisition conditions were carefully optimized by using 8-fold frame averaging, an oil immersion objective (100×, NA 1.3; or 40×, NA 1.0), and a pinhole size of 2.5 optical units. Under these experimental conditions, lateral and axial resolutions of 0.27 and 0.49 µm, respectively, are obtained (Kubitscheck and Peters, 1998Kubitscheck U. Peters R. Localization of single nuclear pore complexes by confocal laser scanning microscopy and analysis of their distribution.Meth Cell Biol. 1998; 53: 79-98Crossref PubMed Google Scholar). Care was taken to exploit the 8 bit dynamic range of the instrument fully. Laser power was adjusted such that no significant photobleaching occurred during the experiments. HDF were plated at a density of 0.5 × 106 per ml into 100 mm Petri dishes in complete medium. They were switched to RPMI 1640 containing 2% FBS 24 h prior to treatment. Phorbol myristate acetate (PMA, Sigma) or recombinant human IL-1β (Roche Diagnostics, Mannheim, Germany) were added to a final concentration of 10 ng per ml each. For UV irradiation cells were exposed to UVA light at a dose of 15 J per cm2 (Scharffetter et al., 1991Scharffetter K. Wlaschek M. Hogg A. et al.UVA irradiation induces collagenase in human dermal fibroblasts in vitro and in vivo.Arch Dermatol Res. 1991; 283: 506-511Crossref PubMed Scopus (310) Google Scholar;Wlaschek et al., 1994Wlaschek M. Heinen G. Poswig A. Schwarz A. Krieg T. Scharffetter-Kochanek K. UVA-induced autocrine stimulation of fibroblast-derived collagenase/MMP-1 by interrelated loops of interleukin-1 and interleukin-6.Photochem Photobiol. 1994; 59: 550-556Crossref PubMed Scopus (225) Google Scholar). The source of the UV light was a UVA device Sellasol (SellasSunlight, Gevelsberg, Germany), which emits most of its energy within the UVA range (320–400 nm) with an emission peak at 365 nm. During UV exposure, culture medium was replaced by phosphate-buffered saline (PBS) and cells were maintained at 37°C in a water bath. Mock-irradiated cells were subjected to the identical procedure except irradiation. Culture supernatants and cellular extracts were harvested 24 and 48 h after treatment with the indicated stimuli. Supernatants were supplemented with protease inhibitors (10 µg per ml aprotinin, 5 µg per ml leupeptin, 1 mM phenylmethylsulfonyl fluoride), centrifuged at 4500g, and stored at -80°C until use. Cells were harvested into lysis buffer (for α-MSH and ACTH) or PBS (for β-ED) supplemented with protease inhibitors as described above. Supernatants (10 ml) and cellular extracts (1 ml) used for detection of α-MSH were 45- and 5-fold concentrated using C18 columns (Waters, Milford, MA). For detection of α-MSH a commercially available radioimmunoassay was used (Euro-Diagnostica, Malmö, Sweden). The detection limit of this assay was 5 pg per ml with no crossreactivity against des-amido-α-MSH, ACTH 1–13, ACTH 1–24, ACTH 1–39, β-MSH, and γ-MSH. ACTH was measured by an immunoradiometric assay from DiaSorin (Stillwater, MN); the sensitivity was 1.5 pg per ml with crossreactivity of less than 0.1% for ACTH 18–24, ACTH 1–10, β-ED, α-MSH, and β-MSH. β-ED was measured using an enzyme immunoassay from Peninsula Laboratories (Belmont, CA), with a sensitivity of 40 pg per ml and no crossreactivity against α-MSH, ACTH, γ-ED, α-ED, Met-enkephalin, or dynorphin. A standard curve was constructed for each assay. Experiments were performed at least three times. Data were analyzed by the unpaired Student's t test. Samples of normal adult human skin (n = 2) derived from patients undergoing routine surgery for therapeutic or diagnostic reasons were examined. The specimens were fixed and processed as described previously (Böhm et al., 1999aBöhm M. Metze D. Schulte U. Becher E. L" @default.
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