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Expression of Proopiomelanocortin Peptides in Human Dermal Microvascular Endothelial Cells: Evidence for a Regulation by Ultraviolet Light and Interleukin-1

2000; Elsevier BV; Volume: 115; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.2000.00174.x

1523-1747

Thomas Scholzen, Thomas Brzoska, Michaela Fastrich, Meinhard Schiller, Markus Böhm, Thomas Schwarz, Thomas A. Luger, D.-H. Kalden, Cheryl A. Armstrong, John C. Ansel,

Dermatology and Skin Diseases

Proopiomelanocortin peptides such as α-melanocyte-stimulating hormone and adrenocorticotropin are expressed in the epidermal and dermal compartment of the skin after noxious stimuli and are recognized as modulators of immune and inflammatory reactions. Human dermal microvascular endothelial cells mediate leukocyte–endothelial interactions during cutaneous inflammation by the expression of cellular adhesion molecules and cytokines such as interleukin-1. This study addresses the hypothesis that human dermal microvascular endothelial cells express both proopiomelanocortin and prohormone convertases, which are required to generate proopiomelanocortin peptides. Semiquantitative reverse transcriptase polymerase chain reaction and northern blot studies revealed a constitutive expression of proopiomelanocortin mRNA by human dermal microvascular endothelial cells in vitro that was time- and concentration-dependently upregulated by interleukin-1β. Furthermore, irradiation of human dermal microvascular endothelial cells with ultraviolet A1 (30 J per cm2) or ultraviolet B (12.5 mJ per cm2) enhanced proopiomelanocortin expression as well as the production and release of the proopiomelanocortin peptides adrenocorticotropin and α-melanocyte-stimulating hormone. In addition to proopiomelanocortin, prohormone convertase 1 mRNA expression was detected by reverse transcriptase polymerase chain reaction in unstimulated human dermal microvascular endothelial cells and was augmented after exposure to α-melanocyte- stimulating hormone, interleukin-1β, or irradiation with ultraviolet. These findings demonstrate that human dermal microvascular endothelial cells express proopiomelanocortin and prohormone convertase 1 required for the generation of adrenocorticotropin. Additionally, human dermal microvascular endothelial cells express mRNA for the prohormone convertase 2 binding protein 7B2. Taken together these findings indicate that human dermal microvascular endothelial cells upon stimulation express both proopiomelanocortin and prohormone convertases required for the generation of α-melanocyte-stimulating hormone. As proopiomelanocortin peptides were found to regulate the production of human dermal microvascular endothelial cell cytokines and adhesion molecules and to have a variety of anti-inflammatory properties these peptides may significantly contribute to the modulation of skin inflammation. Proopiomelanocortin peptides such as α-melanocyte-stimulating hormone and adrenocorticotropin are expressed in the epidermal and dermal compartment of the skin after noxious stimuli and are recognized as modulators of immune and inflammatory reactions. Human dermal microvascular endothelial cells mediate leukocyte–endothelial interactions during cutaneous inflammation by the expression of cellular adhesion molecules and cytokines such as interleukin-1. This study addresses the hypothesis that human dermal microvascular endothelial cells express both proopiomelanocortin and prohormone convertases, which are required to generate proopiomelanocortin peptides. Semiquantitative reverse transcriptase polymerase chain reaction and northern blot studies revealed a constitutive expression of proopiomelanocortin mRNA by human dermal microvascular endothelial cells in vitro that was time- and concentration-dependently upregulated by interleukin-1β. Furthermore, irradiation of human dermal microvascular endothelial cells with ultraviolet A1 (30 J per cm2) or ultraviolet B (12.5 mJ per cm2) enhanced proopiomelanocortin expression as well as the production and release of the proopiomelanocortin peptides adrenocorticotropin and α-melanocyte-stimulating hormone. In addition to proopiomelanocortin, prohormone convertase 1 mRNA expression was detected by reverse transcriptase polymerase chain reaction in unstimulated human dermal microvascular endothelial cells and was augmented after exposure to α-melanocyte- stimulating hormone, interleukin-1β, or irradiation with ultraviolet. These findings demonstrate that human dermal microvascular endothelial cells express proopiomelanocortin and prohormone convertase 1 required for the generation of adrenocorticotropin. Additionally, human dermal microvascular endothelial cells express mRNA for the prohormone convertase 2 binding protein 7B2. Taken together these findings indicate that human dermal microvascular endothelial cells upon stimulation express both proopiomelanocortin and prohormone convertases required for the generation of α-melanocyte-stimulating hormone. As proopiomelanocortin peptides were found to regulate the production of human dermal microvascular endothelial cell cytokines and adhesion molecules and to have a variety of anti-inflammatory properties these peptides may significantly contribute to the modulation of skin inflammation. β-lipotrophic hormone human dermal microvascular endothelial cell melanocortin receptor melanocyte stimulating hormone prohormone convertase proopiomelanocortin Proopiomelanocortin (POMC) peptides, originally discovered as pituitary hormones, have been detected in various tissues including the skin and are expressed by epidermal and dermal cells such as melanocytes, keratinocytes, or fibroblasts as well as by inflammatory cells including cutaneous monocytes, macrophages, and neutrophils (reviewed byLuger et al., 1997Luger T.A. Scholzen T. Grabbe S. The role of α-melanocyte-stimulating hormone in cutaneous biology.J Invest Dermatol Symp Proceedings of The. 1997; 2: 87-93Abstract Full Text PDF PubMed Scopus (127) Google Scholar;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: 1020-1979Google Scholar). In these cells, transcription and release of POMC peptides in vivo and in vitro changes during the hair cycle (Slominski et al., 1992Slominski A. Paus R. Mazurkiewicz J. Proopiomelanocortin expression in the skin during induced hair growth in mice.Experientia. 1992; 48: 50-54Crossref PubMed Scopus (102) Google Scholar,Slominski et al., 1993aSlominski A. Paus R. Wortsman J. On the potential role of proopiomelanocortin in skin physiology and pathology.Mol Cell Endocrinol. 1993; 93: C1-C6Crossref PubMed Scopus (124) Google Scholar) and is increased after trauma, infection (Catania et al., 1994Catania A. Manfredi M.G. Airaghi L. Ceriani G. Gandino A. Lipton J.M. Cytokine antagonists in infectious and inflammatory disorders.Ann N Y Acad Sci. 1994; 741: 149-161Crossref PubMed Scopus (16) Google Scholar,Catania et al., 1998Catania A. Airaghi L. Garofalo L. Cutuli M. Lipton J.M. The neuropeptide alpha-MSH in HIV infection and other disorders in humans.Ann N Y Acad Sci. 1998; 840: 848-856Crossref PubMed Scopus (29) Google Scholar), or exposure to ultraviolet (UV) light. This may in part be secondary to the release of interleukin-1 (IL-1), which is capable of enhancing POMC production (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 (310) Google Scholar;Chakraborty et al., 1995Chakraborty A. Slominski A. Ermak G. Hwang J. Pawelek J. Ultraviolet B and melanocyte-stimulating hormone (MSH) stimulate mRNA production for alpha MSH receptors and proopiomelanocortin-derived peptides in mouse melanoma cells and transformed keratinocytes.J Invest Dermatol. 1995; 105: 655-659Crossref PubMed Scopus (104) Google Scholar,Chakraborty et al., 1996aChakraborty A.K. Funasaka Y. Slominski A. Ermak G. Hwang J. Pawelek J.M. Ichihashi M. Production and release of proopiomelanocortin (POMC) derived peptides by human melanocytes and keratinocytes in culture: regulation by ultraviolet B.Biochim Biophys Acta. 1996; 1313: 130-138Crossref PubMed Scopus (232) Google Scholar;Wintzen et al., 1996Wintzen M. Yaar M. Burbach J.P. Gilchrest B.A. Proopiomelanocortin gene product regulation in keratinocytes.J Invest Dermatol. 1996; 106: 673-678Crossref PubMed Scopus (122) Google Scholar). In neuroendocrine tissues, post-translational processing of an inactive cytoplasmic POMC prohormone generates up to eight different POMC peptides including α-, β-, γ-melanocyte-stimulating hormone (MSH), adrenocorticotrophic hormone (ACTH), and β-endorphin. This generation involves proteolytic cleavage of the POMC precursor protein by prohormone convertases (PC) 1 and 2, which belong to the subtilisin/kexin-type, as well as α-amidation or acetylation (Marcinkiewicz et al., 1993Marcinkiewicz M. Day R. Seidah N.G. Chretien M. Ontogeny of the prohormone convertases PC1 and PC2 in the mouse hypophysis and their colocalization with corticotropin and alpha-melanotropin.Proc Natl Acad Sci U S A. 1993; 90: 4922-4926Crossref PubMed Scopus (126) 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). In skin cells, POMC expression can be induced by UV light, which has been implicated in cutaneous carcinogenesis and local as well as systemic immunosuppression (Kripke, 1990Kripke M.L. Effects of UV radiation on tumor immunity.J Natl Cancer Inst. 1990; 82: 1392-1396Crossref PubMed Scopus (79) Google Scholar;Shreedhar et al., 1998Shreedhar V. Giese T. Sung V.W. Ullrich S.E.A. cytokine cascade including prostaglandin E2, IL-4 and IL-10 is responsible for UV-induced systemic immune suppression.J Immunol. 1998; 160: 3783-3789PubMed Google Scholar;Slominski and Pawelek, 1998Slominski A. Pawelek J. Animals under the sun: effects of ultraviolet radiation on mammalian skin.Clin Dermatol. 1998; 16: 503-515Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar;Luger et al., 1999Luger T.A. Schwarz T. Kalden D.-H. Scholzen T.E. Schwarz A. Brzoska T. Role of epidermal cell-derived α-melanocyte stimulating hormone in ultraviolet light mediated local immunosuppression.Ann N Y Acad Sci. 1999; 885: 209-216Crossref PubMed Scopus (40) Google Scholar). The latter has been attributed to impaired antigen-presenting functions of Langerhans cells and the induction of anti-inflammatory mediators such as IL-10 and α-MSH or, in some cases, calcitonin gene-related peptide (Niizeki and Streilein, 1997Niizeki H. Streilein J.W. Hapten-specific tolerance induced by acute, low-dose ultraviolet B radiation of skin is mediated via interleukin-10.J Invest Dermatol. 1997; 109: 25-30Crossref PubMed Scopus (70) Google Scholar;Luger et al., 1998Luger T.A. Scholzen T. Brzoska T. Becher E. Slominski A. Paus R. Cutaneous immunomodulation and coordination of skin stress responses by alpha-melanocyte-stimulating hormone.Ann N Y Acad Sci. 1998; 840: 381-394Crossref PubMed Scopus (97) Google Scholar;Scholzen et al., 1999Scholzen T.E. Brzoska T. Kalden D.-H. O'Reilly F.M. Armstrong C.A. Luger T.A. Ansel J.C. Effect of ultraviolet light on release of neuropeptides and neuroendocrine hormones in the skin: mediators of photodermatitis and cutaneous inflammation.J Invest Dermatol Symp Proceedings of The. 1999; 4: 55-60Abstract Full Text PDF PubMed Scopus (70) Google Scholar). Among POMC peptides, α-MSH is regarded as a neurohormone with extensive immunomodulatory capacities (Luger et al., 1998Luger T.A. Scholzen T. Brzoska T. Becher E. Slominski A. Paus R. Cutaneous immunomodulation and coordination of skin stress responses by alpha-melanocyte-stimulating hormone.Ann N Y Acad Sci. 1998; 840: 381-394Crossref PubMed Scopus (97) Google Scholar). α-MSH has been demonstrated to regulate proliferation and differentiation of keratinocytes and melanocytes and to modulate fibroblast and endothelial cell cytokine production in vitro (Lipton and Catania, 1997Lipton J.M. Catania A. Anti-inflammatory actions of the neuroimmunomodulator alpha-MSH.Immunol Today. 1997; 18: 140-145Abstract Full Text PDF PubMed Scopus (358) Google Scholar;Luger et al., 1998Luger T.A. Scholzen T. Brzoska T. Becher E. Slominski A. Paus R. Cutaneous immunomodulation and coordination of skin stress responses by alpha-melanocyte-stimulating hormone.Ann N Y Acad Sci. 1998; 840: 381-394Crossref PubMed Scopus (97) Google Scholar). It is capable of downregulating the expression of the costimulatory molecules CD86 and CD40 on monocytes and peripheral-blood-derived dendritic cells. In addition, it induces monocyte anti-inflammatory cytokines such as IL-10 in vitro (Bhardwaj et al., 1996Bhardwaj R.S. Schwarz A. Becher E. Mahnke K. Aragane Y. Schwarz T. Luger T.A. Pro-opiomelanocortin-derived peptides induce IL-10 production in human monocytes.J Immunol. 1996; 156: 2517-2521PubMed Google Scholar, 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 alpha-melanocyte-stimulating hormone receptor MC-1 on human monocytes.J Immunol. 1997; 158: 3378-3384PubMed 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). In vivo, α-MSH demonstrates immunosuppressive activities both locally and systemically, such as the inhibition of murine contact hypersensitivity and the induction of hapten-specific tolerance (Grabbe et al., 1996Grabbe S. Bhardwaj R.S. Mahnke K. Simon M.M. Schwarz T. Luger T.A. Alpha-melanocyte-stimulating hormone induces hapten-specific tolerance in mice.J Immunol. 1996; 156: 473-478PubMed Google Scholar;Lipton and Catania, 1997Lipton J.M. Catania A. Anti-inflammatory actions of the neuroimmunomodulator alpha-MSH.Immunol Today. 1997; 18: 140-145Abstract Full Text PDF PubMed Scopus (358) Google Scholar). POMC peptide effects are exerted by activation of 5 G-protein-coupled melanocortin receptors (MC-1R–MC-5R) (Cone et al., 1996Cone R.D. Lu D. Koppula S. et al.The melanocortin receptors: agonists, antagonists, and the hormonal control of pigmentation.Recent Prog Horm Res. 1996; 51: 287-317PubMed Google Scholar). Epidermal and dermal cells predominantly express MC-1R, which exhibits high affinity for α-MSH and ACTH. Recently, human dermal microvascular endothelial cells (HDMECs) were found to express MC-1R. Functionally, MC-1R-expressing HDMECs responded to stimulation with α-MSH with an increased production of the C-X-C chemokines IL-8 and growth-related oncogene α(Hartmeyer et al., 1930–37, 1997Hartmeyer M. Scholzen T. Becher E. Bhardwaj R.S. Schwarz T. Luger T.A. Human dermal microvascular endothelial cells express the melanocortin receptor type 1 and produce increased levels of IL-8 upon stimulation with alpha-melanocyte-stimulating hormone.J Immunol. 1997; 159: 1930-1937PubMed Google Scholar;Scholzen et al., 1998aScholzen T. Armstrong C.A. Luger T.A. Bunnett N. Olerud J.E. Ansel J.C. Neuropeptides in the skin: interactions between the neuroendocrine and the skin immune systems.Exp Dermatol. 1998; 7: 81-96Crossref PubMed Scopus (353) Google Scholar). The expression of HDMEC cellular adhesion molecules and cytokines are crucial events that mediate leukocyte–endothelial cell interaction and transmigration into the extravascular tissue during skin inflammation (Swerlick and Lawley, 1993Swerlick R.A. Lawley T.J. Role of microvascular endothelial cells in inflammation.J Invest Dermatol. 1993; 100: 111S-115SAbstract Full Text PDF PubMed Google Scholar;Barker, 1995Barker J.N. Adhesion molecules in cutaneous inflammation.Ciba Found Symp. 1995; 189: 91-101PubMed Google Scholar). These events require the activation of endothelial cells by proinflammatory cytokines such as IL-1 or tumor necrosis factor α (TNF-α) that are released from epidermal keratinocytes (Luger and Schwarz, 1995Luger T.A. Schwarz T. Effects of UV-light on cytokines and neuroendocrine hormones.in: Krutmann J. Elmers C. Photoimmunology. Oxford, Blackwell1995: 55-76Google Scholar). Endothelial cells are also capable of synthesizing a repertoire of growth factors, cytokines, and chemokines. Increased production of these factors can be observed after exposure of endothelial cells to certain cytokines, neuropeptides, or UV light (Goebeler et al., 1997Goebeler M. Yoshimura T. Toksoy A. Ritter U. Brocker E.B. Gillitzer R. The chemokine repertoire of human dermal microvascular endothelial cells and its regulation by inflammatory cytokines.J Invest Dermatol. 1997; 108: 445-451Crossref PubMed Scopus (97) Google Scholar;Mantovani et al., 1997Mantovani A. Sozzani S. Vecchi A. Introna M. Allavena P. Cytokine activation of endothelial cells: new molecules for an old paradigm.Thromb Haemostas. 1997; 78: 406-414PubMed Google Scholar;Scholzen et al., 1998aScholzen T. Armstrong C.A. Luger T.A. Bunnett N. Olerud J.E. Ansel J.C. Neuropeptides in the skin: interactions between the neuroendocrine and the skin immune systems.Exp Dermatol. 1998; 7: 81-96Crossref PubMed Scopus (353) Google Scholar; 1998b). In order to get further insight into the significance of MC-1R and its ligands for endothelial cell biology we examined the HDMEC capability for expressing POMC and POMC-processing enzymes. In this study we demonstrate that HDMECs synthesize POMC mRNA and release the POMC peptides ACTH and α-MSH, which can be regulated by IL-1 or UV light. HDMECs were isolated from human foreskins by trypsin treatment and Percoll gradient centrifugation using a protocol modified fromKubota et al., 1988Kubota Y. Kleinman H.K. Martin G.R. Lawley T.J. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures.J Cell Biol. 1988; 107: 1589-1598Crossref PubMed Scopus (924) Google Scholar. Briefly, neonatal foreskins were cut into 5 mm stripes, placed in a 100 mm Petri dish, and washed in phosphate-buffered saline (PBS) containing 0.3% trypsin and 0.2% ethylenediamine tetraacetic acid at 4°C. To separate dermis and epidermis, the skin segments were incubated for 18 h with 2.5% trypsin. Subsequently, epidermis and dermis were separated using sterile forceps. The dermal segments were placed in a Petri dish containing 5 ml modified Eagle's medium and microvascular fragments were expressed by compression of individual fragments of dermis with the side of a scalpel blade. The microvascular fragments were passed through a 100 μm nylon mesh and collected. The microvascular segments were layered on a Percoll (Pharmacia, Uppsala, Sweden) gradient preformed by centrifugation of 35% Percoll in Hank's balanced salt solution (HBSS) at 30,000g for 10 min. The gradient was spun at 400g for 15 min at room temperature. The fraction with a density less than 1.048 g per ml containing endothelial cells was then plated in 100 mm Primaria Petri dishes (Falcon Plastics, Cockeysville, MD). Nonattached cells were removed by washing with HBSS. Cells were grown in endothelial cell basal medium (EBM-Kit MV; PromoCell, Heidelberg, Germany) supplemented with 10% fetal bovine serum, 0.1 ng per ml epidermal growth factor, 1.0 ng per ml basic fibroblast growth factor, and 1.0 μg per ml hydrocortisone without antibiotics (growth medium). Typically, cells in passages 3–5 were used for experiments. In order to verify that cultured HDMECs were free of contaminating cells such as fibroblasts HDMEC cultures were characterized by their typical cobblestone morphology using light microscopy and by flow cytometry analysis regarding their capacity to express factor-VIII-like antigen. To analyze POMC or PC expression HDMECs (2 × 106) were grown in 100 mm Petri dishes for 24 h to 80%-90% confluence in growth medium as described above. Subsequently, the cells were cultured for 15 h or overnight in medium containing 2% fetal bovine serum only (depletion medium), and were then treated with α-MSH (Bachem, Heidelberg, Germany) in various concentrations (10-8-10-12 M) or with IL-1β (0.1–10.0 ng per ml; Sigma, St. Louis, MO) in fresh depletion medium for 1–48 h. Endothelial cells (2 × 106) were grown in 100 mm Petri dishes and depleted as described above. For UV irradiation, the culture medium was replaced by PBS and cells were irradiated with a bank of four FS20 fluorescent lamps (Westinghouse Electric, Pittsburgh, PA), which emit most of their energy within the UVB range (280–320 nm) with an emission peak at 313 nm. The UV output measured at 310 nm using an IL 1700 research radiometer was 8.0 W per m2 at a distance of 28 cm. For UVA1 treatment, cells were exposed to a UVASUN 5000 irradiation device (Mutzhas, Munich, Germany) emitting in the range 320–465 nm, with a maximum at 375 nm. The emission was filtered with UVACRYL (Mutzhas) and UG1 (Schott Glaswerke, Munich, Germany) and consisted exclusively of wavelengths greater than 340 nm. Cell viability before and after irradiation was more than 95% as determined by trypan blue exclusion. After irradiation, the PBS was replaced with fresh medium and cells were further incubated for the indicated time. Total RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62307) Google Scholar). The RNA pellets were washed with 80% ethanol, dried, and dissolved in diethylpyrocarbonate-treated RNase-free water. To avoid DNA contamination, total RNA was treated with 10 U RNase-free DNase I (Boehringer Mannheim, Germany) in a buffer containing 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH 8.3 for 1 h at 37°C. One microgram RNA was subjected to reverse transcription using the Promega Reverse Transcription System in a final volume of 20 μl containing 5 mM MgCl2, 1 × reverse transcriptase buffer [10 mM Tris-HCI (pH 8.8 at 25°C), 50 mM KCl, 0.1% Triton X-100], 1 mM of each dNTP, 1 U per μl rRNasin, 15 U AMV reverse transcriptase, and 0.5 μg oligo-(dT)15 primer. Tubes were incubated for 60 min at 42°C with a 5 min inactivation of the AMV reverse transcriptase at 95°C, chilled on ice, and diluted to a final volume of 100 μl w/DEPC-H2O. PCR conditions allowing reliable comparison of POMC and PC1 expression with β-actin mRNA as housekeeping gene expression in different samples were established using a protocol modified fromPaludan and Thestrup-Pedersen, 1992Paludan K. Thestrup-Pedersen K. Use of the polymerase chain reaction in quantification of interleukin-8 messenger RNA in minute epidermal samples.J Invest Dermatol. 1992; 99: 830-835Abstract Full Text PDF PubMed Google Scholar) by making serial dilutions of template cDNA at constant cycle numbers for each primer pair to verify that the subsequent PCR reactions were performed in the linear range of PCR amplification. Subsequently, cDNA mixtures were diluted to obtain similar amounts of PCR product specific for amplified β-actin and subjected to amplification of POMC (143 bp) and PC1-specific (674 bp), PC2-specific (191 bp), or 7B2-specific (443 bp) PCR products using the oligonucleotide primer pairs listed below. For PCR amplification, 50 μl reactions containing appropriate volumes of diluted cDNA reaction mix, 200 μM dNTP (each), 20–50 pM of each primer, and the standard buffer supplemented with Taq Polymerase (2.5 U per reaction, Promega) and 1.5–2.0 mM MgCl2 were used. Nucleotide sequences for PCR primers and amplification programs were as follows. β-actin was amplified using the sense primer 1 5′-CAC- CTTCTACAATGAGCTGC-3′ and the antisense primer 1 5′-TTCAT- GAGGTAGTCCGTCAG-3′, or alternatively the β-actin sense primer 2 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′ and the antisense primer 2 5′-CGTCATACTCCTGCTTGCTGATCCACATC- TGC-3′ (Clontech, LaJolla, CA), and the following amplification program: 1 cycle of 94°C, 10 min; 58°C, 2 min; 72°C, 1 min; followed by 33 cycles of 94°C, 45 s; 58°C, 45 s; 72°C, 1 min; and a final cycle of 94°C, 45 s; 58°C, 45 s; and 72°C, 10 min. POMC was amplified using the sense primer 5′-TCAGCCTGCCTGGAAGATGCC-3′, the antisense primer 5′-GGTTGCTTTCCGTGGTGAGGTC-3′, and the following amplification program: 1 cycle of 94°C, 10 min; 64°C, 1 min; 72°C, 1 min; followed by 33 cycles of 94°C, 45 s; 64°C, 45 s; 72°C, 1 min; and a final cycle of 94°C, 45 s; 64°C, 45 s; and 72°C, 10 min. PC1 was amplified using the sense primer 5′-AGCAAACCCAAATCTCACCTG-3′, the antisense primer 5′-TCTCCACCCCTCTTCTGTCAT-3′, and the following amplification program: 1 cycle of 94°C, 10 min; 53°C, 45 s; 72°C, 1 min; followed by 33 cycles of 94°C, 45 s; 53°C, 45 s; 72°C, 1 min; and a final cycle of 94°C, 45 s; 53°C, 45 s; and 72°C, 10 min. PC2 was amplified using the sense primer 5′-GTGAAAATGGCTAAAGACTGG-3′, the antisense primer 5′-GTTGCGTTGACCGTGATGACA-3′, and the following amplification program: 1 cycle of 94°C, 10 min; 52°C, 30 s; 72°C, 45 s; followed by 33 cycles of 94°C, 30 s; 52°C, 30 s; 72°C, 45 s; and a final cycle of 94°C, 30 s; 52°C, 30 s; and 72°C, 10 min. 7B2 was amplified using the sense primer 5′-CACCAGGCCATGAATCTT-3′, the antisense primer 5′-CTG- GATCCTTATCCTCATCTG-3′, and the following amplification program: 1 cycle of 94°C, 5 min; 53°C, 45 s; 72°C, 2 min; followed by 33 cycles of 94°C, 1 min; 53°C, 45 s; 72°C, 1 min; and a final cycle of 94°C, 1 min; 53°C, 45 s; and 72°C, 10 min. Aliquots of reaction products were run on 1.5% agarose gels and analyzed by product size compared with a coamplified control template, or by cutting of the isolated fragment with appropriate restriction enzymes, or by DNA sequencing. To semiquantify the relative amounts of POMC or PC1 mRNA the signal intensity of the POMC or PC1 PCR product was compared by densitometer reading with that of a β-actin PCR product amplified from the same cDNA in a separate PCR reaction. Amplification fragments were separated on 1.5% agarose gels. The intensity of the ethidium-bromide-stained band of a specific product was densitometrically evaluated using a BioProfil Video Densitometer, Image Analysis and Photo Documentation System (CCD video camera/transilluminator connected to a PC equipped with a frame grabber video card) with BioProfil 2-D Image Processing and Analyzing Software (Fröbel Labortechnik, Wasserburg, Germany). Densitometer readings of POMC- or PC1-specific PCR products were normalized to the β-actin product density in the respective sample. Subsequently, the density of POMC or PC1 product amplified from cDNA prepared from stimulated cells was related to that of unstimulated control cells at any given time point. Unless stated otherwise, results from three different experiments were expressed in percentage of control as the mean ± SEM with the density of unstimulated controls set to 100% for each time point analyzed. Total RNA samples (15 μg per lane) were electrophoresed on a 1% agarose formaldehyde gel, transferred to nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ), and hybridized with a 32P-radiolabeled human cDNA probe corresponding to human POMC exon 3 using RapidHybe hybridization solution (Amersham). DNA probes were radiolabeled using the random hexamer method (Feinberg and Vogelstein, 1983Feinberg A.P. Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.Anal Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16456) Google Scholar) (rediprime labeling system; Amersham). Normalization of cytokine mRNA for equal RNA loading of lanes and northern blot transfer efficiency was accomplished by hybridization of filters with a cDNA fragment of human β-actin. Cell supernatants or cell lysates were harvested as described earlier (Chakraborty et al., 1995Chakraborty A. Slominski A. Ermak G. Hwang J. Pawelek J. Ultraviolet B and melanocyte-stimulating hormone (MSH) stimulate mRNA production for alpha MSH receptors and proopiomelanocortin-derived peptides in mouse melanoma cells and transformed keratinocytes.J Invest Dermatol. 1995; 105: 655-659Crossref PubMed Scopus (104) Google Scholar). Briefly, aprotinin (0.01%) and phenylmethylsulfonyl fluoride (1 mM) were added to cell supernatants of UV- or cytokine-treated HDMECs after stimulation. Acetic acid (5 N) was added and media were centrifuged at 16,000g to remove any precipitates. Supernatants were collected, the pH was adjusted to 7.5, and the media were centrifuged again to remove any precipitates. Supernatants were collected, freeze dried under vacuum, and stored at -80°C. To harvest cell lysates, the medium was removed, and the cells were washed with PBS, collected, homogenized in 5 N acetic acid using a cell scraper, and processed as described above. The ACTH or α-MSH contents of freeze-dried cell supernatants or lysates were analyzed using commercially available radioimmunoassay (EuroDiagnostica, Malmö, Sweden). According to the manufacturer's protocol, the antiserum used in the α-MSH radioimmunoassay was directed against the C-terminal part of α-MSH recognizing α-MSH and Des-acetyl-α-MSH, with no cross-reactivity against ACTH, β-MSH, or γ-MSH. The antiserum against ACTH was directed to the N-terminal portion of ACTH 1–39, with no cross-reactivity against α-, β-, or γ-MSH. The total protein contents of cell lysates were determined using the Bio-Rad D/C protein assay system (Bio-Rad Laboratories, Hercules, CA). Unless indicated otherwise, experiments were performed at least three times and are presented as mean ± SEM. The unpaired Student's t test was used to calculate the statistical significance. To determine if HDMECs express POMC mRNA, RT-PCR was conducted using the amplification of a 143 bp POMC fragment that corresponds to POMC exon 2. The amplification of a 300 bp β-actin fragment served as internal control reflecting the relative amount of mRNA in each sample.