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

Expression of Proopiomelanocortin Peptides in Human Dermal Microvascular Endothelial Cells: Evidence for a Regulation by Ultraviolet Light and Interleukin-1 Thomas E. Scholzen, Dirk-Henner Kalden,* Thomas Brzoska, Michaela Fastrich, Meinhard Schiller, Markus BoÈhm, T. Schwarz, Cheryl A. Armstrong,* John C. Ansel,* and Thomas A. Luger

Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, Department of Dermatology, University of MuÈnster, Germany; *Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia, U.S.A.

Proopiomelanocortin peptides such as a-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 in¯ammatory reactions. Human dermal microvascular endothelial cells mediate leukocyte±endothelial interactions during cutaneous in¯ammation 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 timeand concentration-dependently upregulated by interleukin-1b. 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 a-melanocyte-stimulating hormone. In addition to

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roopiomelanocortin (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 ®broblasts as well as by in¯ammatory cells including cutaneous monocytes, macrophages, and neutrophils Manuscript received May 15, 2000; revised July 12, 2000; accepted for publication September 7, 2000. Reprint requests to: Prof. Dr Thomas A. Luger, Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, Department of Dermatology, University of MuÈnster, Von-Esmarch-Str. 56, 48149 MuÈnster, Germany. Email: [email protected] Abbreviations: b-LPH, b-lipotrophic hormone; HDMEC, human dermal microvascular endothelial cell; MC-R, melanocortin receptor; MSH, melanocyte stimulating hormone; PC, prohormone convertase; POMC, proopiomelanocortin. 0022-202X/00/$15.00

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 a-melanocytestimulating hormone, interleukin-1b, or irradiation with ultraviolet. These ®ndings 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 ®ndings indicate that human dermal microvascular endothelial cells upon stimulation express both proopiomelanocortin and prohormone convertases required for the generation of a-melanocytestimulating 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-in¯ammatory properties these peptides may signi®cantly contribute to the modulation of skin in¯ammation. Key words: endothelial cell/melanocytestimulating hormone/prohormone convertase/proopiomelanocortin/UV light. J Invest Dermatol 115:1021±1028, 2000

(reviewed by Luger et al, 1997; Slominski et al, 2000). In these cells, transcription and release of POMC peptides in vivo and in vitro changes during the hair cycle (Slominski et al, 1992, 1993a) and is increased after trauma, infection (Catania et al, 1994, 1998), 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, 1994; Chakraborty et al, 1995, 1996a; Wintzen et al, 1996). In neuroendocrine tissues, post-translational processing of an inactive cytoplasmic POMC prohormone generates up to eight different POMC peptides including a-, b-, gmelanocyte-stimulating hormone (MSH), adrenocorticotrophic hormone (ACTH), and b-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 a-amidation or acetylation (Marcinkiewicz et al, 1993; Seidah et al, 1999). In skin cells, POMC expression can be induced by

´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 1021

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UV light, which has been implicated in cutaneous carcinogenesis and local as well as systemic immunosuppression (Kripke, 1990; Shreedhar et al, 1998; Slominski and Pawelek, 1998; Luger et al, 1999). The latter has been attributed to impaired antigen-presenting functions of Langerhans cells and the induction of anti-in¯ammatory mediators such as IL-10 and a-MSH or, in some cases, calcitonin gene-related peptide (Niizeki and Streilein, 1997; Luger et al, 1998; Scholzen et al, 1999). Among POMC peptides, a-MSH is regarded as a neurohormone with extensive immunomodulatory capacities (Luger et al, 1998). a-MSH has been demonstrated to regulate proliferation and differentiation of keratinocytes and melanocytes and to modulate ®broblast and endothelial cell cytokine production in vitro (Lipton et al, 1997; Luger et al, 1998). 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-in¯ammatory cytokines such as IL10 in vitro (Bhardwaj et al, 1996, 1997; Becher et al, 1999). In vivo, a-MSH demonstrates immunosuppressive activities both locally and systemically, such as the inhibition of murine contact hypersensitivity and the induction of hapten-speci®c tolerance (Grabbe et al, 1996; Lipton and Catania, 1997). POMC peptide effects are exerted by activation of 5 G-proteincoupled melanocortin receptors (MC-1R±MC-5R) (Cone et al, 1996). Epidermal and dermal cells predominantly express MC-1R, which exhibits high af®nity for a-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 a-MSH with an increased production of the C-X-C chemokines IL-8 and growth-related oncogene a (Hartmeyer et al, 1997; Scholzen et al, 1998a). 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 in¯ammation (Swerlick and Lawley, 1993; Barker, 1995). These events require the activation of endothelial cells by proin¯ammatory cytokines such as IL-1 or tumor necrosis factor a (TNF-a) that are released from epidermal keratinocytes (Luger and Schwarz, 1995). 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, 1997; Mantovani et al, 1997; Scholzen et al, 1998a; 1998b). In order to get further insight into the signi®cance 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 aMSH, which can be regulated by IL-1 or UV light. MATERIALS AND METHODS Cell culture and reagents HDMECs were isolated from human foreskins by trypsin treatment and Percoll gradient centrifugation using a protocol modi®ed from Kubota et al (1988). Brie¯y, 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 modi®ed 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 mm 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).

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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 ®broblast growth factor, and 1.0 mg 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 ®broblasts HDMEC cultures were characterized by their typical cobblestone morphology using light microscopy and by ¯ow cytometry analysis regarding their capacity to express factor-VIII-like antigen. To analyze POMC or PC expression HDMECs (2 3 106) were grown in 100 mm Petri dishes for 24 h to 80%±90% con¯uence 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 a-MSH (Bachem, Heidelberg, Germany) in various concentrations (10±8±10±12 M) or with IL-1b (0.1± 10.0 ng per ml; Sigma, St. Louis, MO) in fresh depletion medium for 1± 48 h. UV treatment Endothelial cells (2 3 106) 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 ¯uorescent 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 ®ltered 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. RNA isolation Total RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). 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. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) One microgram RNA was subjected to reverse transcription using the Promega Reverse Transcription System in a ®nal volume of 20 ml containing 5 mM MgCl2, 1 3 reverse transcriptase buffer [10 mM TrisHCI (pH 8.8 at 25°C), 50 mM KCl, 0.1% Triton X-100], 1 mM of each dNTP, 1 U per ml rRNasin, 15 U AMV reverse transcriptase, and 0.5 mg 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 ®nal volume of 100 ml w/DEPC-H2O. PCR conditions allowing reliable comparison of POMC and PC1 expression with b-actin mRNA as housekeeping gene expression in different samples were established using a protocol modi®ed from Paludan and Thestruppedersen (1992) 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 ampli®cation. Subsequently, cDNA mixtures were diluted to obtain similar amounts of PCR product speci®c for ampli®ed b-actin and subjected to ampli®cation of POMC (143 bp) and PC1-speci®c (674 bp), PC2-speci®c (191 bp), or 7B2-speci®c (443 bp) PCR products using the oligonucleotide primer pairs listed below. For PCR ampli®cation, 50 ml reactions containing appropriate volumes of diluted cDNA reaction mix, 200 mM 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 ampli®cation programs were as follows. b-actin was ampli®ed using the sense primer 1 5¢-CACCTTCTACAATGAGCTGC-3¢ and the antisense primer 1 5¢-TTCATGAGGTAGTCCGTCAG-3¢, or alternatively the b-actin sense primer 2 5¢-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3¢ and the antisense primer 2 5¢-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3¢ (Clontech, LaJolla, CA), and the following ampli®cation 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 ®nal cycle of 94°C, 45 s; 58°C, 45 s; and 72°C, 10 min. POMC was ampli®ed using the sense primer 5¢-TCAGCCTGCCTGGAAGATGCC-3¢, the antisense primer 5¢-

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Figure 1. Kinetics and concentration dependence of POMC mRNA upregulation in relation to b-actin expression after stimulation with IL1b. Semiquantitative RT-PCR using POMC- and b-actin-speci®c primer pairs. HDMECs (2 3 106 cells) were left untreated (control) or stimulated with 0.1 ng per ml or 1 ng per ml IL-1b for the indicated periods of time. Total RNA was harvested and subjected to RT-PCR. Ampli®cation products of POMC (143 bp) and b-actin cDNA (300 bp) were separated on agarose gels (A) and subjected to densitometric evaluation to semiquantify POMC mRNA expression (B). Results are expressed as mean 6 SEM of three individual experiments. *p < 0.05; **p < 0.025; ***p < 0.005 vs control. GGTTGCTTTCCGTGGTGAGGTC-3¢, and the following ampli®cation 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 ®nal cycle of 94°C, 45 s; 64°C, 45 s; and 72°C, 10 min. PC1 was ampli®ed using the sense primer 5¢-AGCAAACCCAAATCTCACCTG-3¢, the antisense primer 5¢TCTCCACCCCTCTTCTGTCAT-3¢, and the following ampli®cation 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 ®nal cycle of 94°C, 45 s; 53°C, 45 s; and 72°C, 10 min. PC2 was ampli®ed using the sense primer 5¢-GTGAAAATGGCTAAAGACTGG-3¢, the antisense primer 5¢GTTGCGTTGACCGTGATGACA-3¢, and the following ampli®cation 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 ®nal cycle of 94°C, 30 s; 52°C, 30 s; and 72°C, 10 min. 7B2 was ampli®ed using the sense primer 5¢-CACCAGGCCATGAATCTT-3¢, the antisense primer 5¢-CTGGATCCTTATCCTCATCTG-3¢, and the following ampli®cation 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 ®nal 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 coampli®ed control template, or by cutting of the isolated fragment with appropriate restriction enzymes, or by DNA sequencing. Quanti®cation of PCR products 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 bactin PCR product ampli®ed from the same cDNA in a separate PCR reaction. Ampli®cation fragments were separated on 1.5% agarose gels. The intensity of the ethidium-bromide-stained band of a speci®c product was densitometrically evaluated using a BioPro®l Video Densitometer, Image Analysis and Photo Documentation System (CCD video camera/ transilluminator connected to a PC equipped with a frame grabber video card) with BioPro®l 2-D Image Processing and Analyzing Software (FroÈbel Labortechnik, Wasserburg, Germany). Densitometer readings of POMCor PC1-speci®c PCR products were normalized to the b-actin product density in the respective sample. Subsequently, the density of POMC or PC1 product ampli®ed 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 6 SEM with the density of unstimulated controls set to 100% for each time point analyzed. Northern hybridization Total RNA samples (15 mg 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, 1983) (rediprime labeling system; Amersham). Normalization of cytokine mRNA for equal RNA loading of lanes and northern blot transfer ef®ciency was accomplished by hybridization of ®lters with a cDNA fragment of human b-actin. ACTH/a-MSH radioimmunoassay Cell supernatants or cell lysates were harvested as described earlier (Chakraborty et al, 1995). Brie¯y, aprotinin (0.01%) and phenylmethylsulfonyl ¯uoride (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 a-MSH contents of freeze-dried cell supernatants or lysates were analyzed using commercially available radioimmunoassay (EuroDiagnostica, MalmoÈ, Sweden). According to the manufacturer's protocol, the antiserum used in the a-MSH radioimmunoassay was directed against the C-terminal part of a-MSH recognizing a-MSH and Des-acetyl-a-MSH, with no crossreactivity against ACTH, b-MSH, or g-MSH. The antiserum against ACTH was directed to the N-terminal portion of ACTH 1±39, with no cross-reactivity against a-, b-, or g-MSH. The total protein contents of cell lysates were determined using the Bio-Rad D/C protein assay system (BioRad Laboratories, Hercules, CA). Statistical signi®cance Unless indicated otherwise, experiments were performed at least three times and are presented as mean 6 SEM. The unpaired Student's t test was used to calculate the statistical signi®cance.

RESULTS HDMECs express POMC mRNA constitutively and upon stimulation with IL-1b To determine if HDMECs express POMC mRNA, RT-PCR was conducted using the ampli®cation of a 143 bp POMC fragment that corresponds to POMC exon 2. The ampli®cation of a 300 bp b-actin fragment served as internal control re¯ecting the relative amount of mRNA in each sample. HDMECs constitutively express low amounts of POMC mRNA at 1, 5, 12, and 24 h (Fig 1A). Previously, it has been demonstrated for keratinocytes in vitro that IL-1b is capable of upregulating POMC mRNA (Schauer et al, 1994). Thus, HDMECs were stimulated with various concentrations of IL-1b and POMC mRNA expression was measured 1, 5, 12, and 24 h post induction (Fig 1A). Semiquantitative densitometric evaluation of PCR products revealed that IL-1b in a concentration-dependent manner upregulated POMC mRNA 1 h after stimulation. Maximum POMC levels were observed 5 h after stimulation with 1 ng per ml IL-1b with a subsequent decline to basal levels of POMC mRNA expression after 24 h (Fig 1B). POMC mRNA expression in HDMECs is upregulated upon UVB or UVA1 irradiation UV irradiation is one of the major stimuli for POMC mRNA and peptide expression in epidermal cells such as melanocytes and keratinocytes (Chakraborty et al, 1995; Slominski and Pawelek, 1998; Luger et al, 1999). We tested the possibility that UV may also have a direct impact on HDMEC POMC production. In unirradiated control cells, the level of POMC mRNA was similar at all time points. Irradiation of HDMECs with UVA1 light (30 J per cm2) increased the amount of POMC mRNA with a 2.5±4-fold induction compared with unirradiated controls at 3 and 48 h post irradiation, respectively (Fig 2A, B). This late inductive effect may be mediated by the autocrine effect of UV-induced HDMEC cytokines. Likewise, irradiation with UVB (12.5 mJ per cm2) resulted in a very similar temporal expression pro®le compared with unstimulated control cells with increased POMC mRNA 1±3 h after irradiation peaking again after 24 and 48 h (Fig 2C, D). In addition, we performed northern hybridization to exclude the unlikely possibility that HDMEC primary cultures were contaminated with few nonendothelial skin cells resulting in false positive RT-PCR results. As evident from Fig 2(E), HDMEC POMC mRNA is induced 3 h after treatment with IL-1 (1 ng per ml), UVA1 light

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Figure 2. Kinetics of POMC mRNA upregulation in relation to bactin expression after stimulation with UV light. HDMECs (2 3 106 cells) were stimulated with UVA1 light (30 J per cm2, A), UVB light (12.5 mJ per cm2, C), or left untreated (control). Total RNA was harvested after the indicated periods of time and subjected to RT-PCR (A, C). Ampli®cation products of POMC (143 bp) and b-actin cDNA (838 bp) were separated on agarose gels and analyzed by densitometry (B, D). Representative data of three different experiments are shown. For northern hybridization (E), HDMECs (2 3 106 cells) were stimulated with UVB light (12.5 mJ per cm2), UVA1 light (30 J per cm2), IL-1b (1 ng per ml), or left untreated (control). Total RNA was harvested after 3 h, and 15 mg per lane were separated on a 1% formaldehyde agarose gel, blotted on nylon membrane, and hybridized with a probe for POMC or b-actin, respectively.

(30 J per cm2), or UVB light (12.5 mJ per cm2). The apparent size of the POMC transcript detected in HDMECs was about 1.1 kB. Viability of UVB- or UVA1-irradiated HDMECs was more than 95% as demonstrated by trypan blue exclusion (data not shown). UV irradiation increases the production of ACTH and aMSH In order to investigate whether UV-induced expression of POMC mRNA is accompanied by an increased accumulation and release of POMC peptides, HDMEC lysates were subjected to analysis using ACTH-speci®c radioimmunoassays after increasing amounts of UV (Fig 3). UVB- or UVA1-treated HDMECs responded to irradiation with an enhanced intracellular level of ACTH 12 h after stimulation compared with untreated controls, which declined to a minimum after 24 h and was slightly elevated again 48 h post irradiation. The induction of ACTH was also dose dependent with doses of 20 mJ per cm2 UVB or 30 J per cm2 UVA1 being most effective. In the supernatants of UVA1-irradiated HDMECs basal ACTH release was increased as early as 1 h with a maximum 24 h after irradiation with UVA1 (Fig 4). Similar data were obtained when HDMECs were treated with UVB (data not shown). HDMEC supernatants also contained low levels of a-MSH after 24 and 48 h that were signi®cantly increased upon irradiation with UVA (30 J per cm2) or UVB (12.5 mJ per cm2) (Fig 5).

HDMECs express PC1 and 7B2 In neuroendocrine cells, the post-translational processing of the POMC prohormone by PC1 generates ACTH and b-lipotrophic hormone (b-LPH), whereas processing by PC2 produces a-, b, and g-MSH and endorphins. As HDMECs release ACTH as well as a-MSH, we further tested, by semiquantitative RT-PCR using primer pairs speci®c for PC1 and PC2, whether HDMECs express PCs necessary for POMC precursor processing. In addition, we examined whether HDMECs express the PC2-binding protein 7B2 that is required for the zymogen activation of PC2. Accordingly, unstimulated cells were found to constitutively express low levels of PC1 transcripts. PC1 mRNA levels in HDMECs were enhanced up to 400% compared with untreated control cells 1±5 h after stimulation with IL-1b (1 ng per ml) or a-MSH (10±8 M, Fig 6A, B). Like the POMC mRNA expression, the expression of PC1 mRNA is also differentially modulated by UVA1 (Fig 7A, B) or UVB light (data not shown). Time course studies of PC1 mRNA expression in UVA1-irradiated HDMECs again revealed a biphasic induction pro®le with an elevated PC1 mRNA level 1±3 h and 24±48 h post irradiation. This suggests that UV affects the expression of PC mRNA in a direct as well as an indirect manner. The level of PC1 mRNA in unirradiated controls was unaffected at all time points. In addition, unstimulated and IL-1b-stimulated HDMECs (Fig 8) as well as transformed dermal microvascular endothelial cells

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Figure 3. Kinetics of intracellular ACTH production after UV. HDMECs were treated with 30 J per cm2 UVA1 or 10 mJ per cm2 UVB as indicated, or left untreated (control), and cells were harvested after 12, 24, and 48 h. ACTH was determined by radioimmunoassay in relation to the total protein contents of cell lysates as determined by BCA assay. Values are given in pmol per g total protein 6 SEM. *p < 0.05; **p < 0.025; ***p < 0.005 vs control at each given time point.

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Figure 4. ACTH release by UV-irradiated HDMECs. Cells were irradiated with 30 J per cm2 UVA1 or left untreated (control) and supernatants were collected after the indicated time periods. ACTH was determined using speci®c radioimmunoassay or enzyme immunoassays. Values are given in pM 6 SEM. *p < 0.05; **p < 0.01; ***p < 0.001 vs control.

(HMEC-1) (data not shown) express mRNA for 7B2 as detected by RT-PCR. Both HMEC-1 and HDMECs also expressed 7B2 protein as detected by western blotting (data not shown). DISCUSSION The interaction between the immune system and neuropeptides that are generated from the POMC prohormone representing a conspicuous part of the neuroendocrine system has been receiving increasing attention. Among the POMC peptides, numerous studies demonstrated a unique physiologic signi®cance as immunomodulator especially for a-MSH in the immune system and the skin immune system in particular (Lipton et al, 1997; Luger et al, 1997; Scholzen et al, 1998a). Recent studies revealed that the skin is not only the target but also the site of POMC expression. It is now well established that normal murine or human keratinocytes, melanocytes, Langerhans cells, and ®broblasts express POMC mRNA and peptides such as a-MSH, ACTH, or b-endorphin in vitro (Schauer et al, 1994; Chakraborty et al, 1995; Farooqui et al, 1995; Wintzen et al, 1996; Teofoli et al, 1997; Peters et al, 2000). Most of these peptides have also been identi®ed by immunohistochemical staining or the combination of peptide extraction, reversed phase high performance liquid chromatography, and radioimmunoassay in murine or human skin in vivo (Thody et al, 1983; Liu and Johansson, 1995; Slominski et al, 1995; 1999; Furkert et al, 1997; Wakamatsu et al, 1997; Slominski and Pawelek, 1998). In this study we demonstrate that POMC mRNA, certain components of the POMC prohormone-processing apparatus, as well as the POMC peptides ACTH and a-MSH are also expressed and released by HDMECs. In addition, it is noteworthy that similar POMC expression and regulation patterns could also be detected in HMEC-1 (Scholzen et al, unpublished). Thus, it appears to be unlikely that our observations on HDMEC POMC expression are due to cell culture contamination with nonendothelial cells such as ®broblasts. Our northern hybridization and semiquantitative RT-PCR studies detected a low expression level of POMC mRNA in unstimulated HDMECs that was signi®cantly enhanced upon stimulation with IL-1 in a time- and concentration-dependent manner. These ®ndings are in accordance with previous studies that demonstrated only minor amounts of POMC transcripts and peptides in unstimulated keratinocytes. Treatment of these cells with IL-1b or UVB signi®cantly upregulated POMC mRNA expression as well as a-MSH and ACTH release (Kock et al, 1991; Schauer et al, 1994; Luger et al, 1999). Likewise, POMC expression in normal skin could only be detected in skin appendages whereas a

Figure 5. a-MSH release by UV-irradiated HDMECs. Cells were irradiated with 30 J per cm2 UVA1, with UVB (12.5 mJ per cm2), or left untreated (control), and supernatants were collected after the indicated time periods. a-MSH was determined using speci®c radioimmunoassay or enzyme immunoassays. Values are given in pM 6 SEM. *p < 0.05; **p < 0.01; ***p < 0.001 vs control.

strong expression of POMC was apparent in lesional skin affected by diseases such as psoriasis or in cutaneous tumors, i.e., in basal cell carcinoma or in melanoma (Slominski et al, 1993b, 1995; Nagahama et al, 1998). Irradiation with UVB or UVA1 in a time- and dose-dependent manner upregulates the POMC mRNA expression and the POMC peptide release by HDMECs. The amount of intracellular as well as released a-MSH and ACTH after UV was comparable with amounts previously reported for keratinocytes and melanocytes (Schauer et al, 1994; Chakraborty et al, 1996b). Notably, the baseline level of a-MSH detected in HDMEC supernatants (around 10 pM = 16.6 pg per ml) was very similar to plasma aMSH levels detected in normal healthy volunteers (12 pM = 20 pg per ml; Catania et al, 1998). In a similar manner to POMC, the mRNA expression of PC 1 is regulated by UV light. This biphasic effect of UV on POMC and PC1 mRNA expression is re¯ected by an initial induction period 1±3 h after irradiation, followed by a decline at 6 and 16 h, and a subsequent induction at 48 h. A comparable biphasic modulation has been reported for TNF-a-induced keratinocyte intercellular adhesion molecule I (ICAM-1) expression as well as for UV-

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Figure 6. Kinetics of PC1 mRNA expression. PC1 mRNA was compared to b-actin expression after stimulation with IL-1b and a-MSH. HDMECs (2 3 106 cells) were left untreated (control) or stimulated with IL-1b (1 ng per ml) or a-MSH (10±8 M), respectively, for the indicated periods of time. Total RNA was subjected to RT-PCR and ampli®cation products of PC1 (674 bp) and b-actin cDNA (300 bp) were separated on agarose gels (A), and evaluated densitometrically to semiquantify PC1 mRNA expression (B). Results are expressed as mean 6 SEM of three individual experiments. *p < 0.05; **p < 0.025; ***p < 0.005 vs control.

Figure 7. Kinetics of PC1 mRNA upregulation in comparison to bactin expression after irradiation with UVA1 light. HDMECs (2 3 106 cells) were left untreated (control) or stimulated with 30 J per cm2 UVA1 for the indicated periods of time. After RT-PCR of total RNA, ampli®cation products of PC1 (674 bp) and b-actin cDNA (834 bp) were separated on agarose gels (A) and subjected to densitometric evaluation to semiquantify PC1 mRNA expression (B). Representative data of two different experiments are shown.

induced keratinocyte TNF-receptor I expression, melanocyte ICAM-1 expression, and for endothelial cell cytokine production (Kirnbauer et al, 1992; Trefzer et al, 1993; Scholzen et al, 1998b). The transcriptional upregulation of POMC and PC1 mRNA by UV light as soon as 1 h post irradiation seems to favor a direct effect of UV on HDMECs indicating that the induction of both genes may represent a part of the immediate-early gene response of HDMECs to UV light. Although UV directly as well as indirectly affects the function of endothelial cells in vitro, there is some concern about the in vivo relevance of these ®ndings as epidermal cells are regarded as the primary target for UV irradiation. There is recent evidence, however, that UVB in the range between 0.5 and 3 minimal erythema doses signi®cantly damages basal keratinocytes as

demonstrated by thymidine dimer formation (Young et al, 1997). In addition, based on transmission data of ex vivo irradiated human epidermis, about 40% of UVA seems to be able to penetrate to the dermis and to reach cutaneous vessels (Everett et al, 1966). Thus, it appears to be likely that a signi®cant amount of UVB and in particular of long-wavelength UVA reaches the upper dermis and directly alters the function of vessel cells in the dermal papillae in vivo. In accordance with previous observations our data demonstrate that only a limited number of stimuli including UV light are capable of inducing POMC expression in epidermal as well as dermal cells. Among various cytokines, so far only IL-1 and TNF-a have been proven to induce POMC expression in human keratinocytes and some epidermoid carcinoma cell lines or ®broblasts (Schauer et al, 1994; Wintzen et al, 1996; Teofoli et al, 1997). In extension of these data, our ®ndings indicate that IL-1 is also capable of increasing the expression level of PC1 mRNA in a time- and concentration-dependent manner. IL-1 has been reported to be induced in HDMECs and other skin cells such as keratinocytes upon irradiation with UVB and UVA1 light1 (Luger et al, 1995; Scholzen et al, 1998b). Thus, it can be speculated that the second induction of POMC and of PC1 mRNA observed in HDMECs after 24 h might be an autocrine or paracrine effect mediated by UV-induced IL-1. It is noteworthy that induction of keratinocyte POMC expression by UVB light in vitro could be blocked by preincubation of keratinocytes with the IL-1 receptor antagonist (Brzoska et al, unpublished observation). Therefore, it seems to be conceivable that, in addition to a direct POMC induction by UV light and an autocrine/paracrine induction of POMC via endothelial-cell-derived IL-1, keratinocyte-derived IL1 may also account for POMC production in endothelial cells in vivo. Understanding the regulation of POMC expression in skin and other tissues in relation to PC expression is important, as the expression level of these proteolytic enzymes may affect the POMC prohormone processing. In the pituitary, POMC expression and post-translational POMC prohormone processing occur in a celland tissue-speci®c manner. In corticotrophic cells of the anterior pituitary, where ACTH and b-LPH are the predominant POMC products, PC1 is highly expressed, whereas only very low levels of PC2 are found. In contrast, intermediate lobe melanotrophic cells express both PC1 and PC2 at high levels with PC2 being more abundant. In this tissue, ACTH and b-LPH are also further processed to a-MSH, corticotrophin-like intermediate lobe peptide, g-LPH, and b-endorphins (Zhou et al, 1993; Castro and Morrison, 1997). A similar system for POMC processing in different skin compartments equivalent to that in melanotrophic and corticotrophic cells of the pituitary has been proposed earlier (Slominski et al, 1993a). Recently, examination of human melanocytes demonstrated that the entire system for POMC processing is present within the melanosome (Peters et al, 2000). 1Scholzen T, Hartmeyer M, Fastrich M, Becher E, Brzoska T, Schwarz T, Luger TA: Chemokine and cytokine expression by human dermal microvascular endothelial cells is differentially modulated upon irradiation with UVA1-light. J Invest Dermatol 108:650, 1997 (abstr.)

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Figure 8. Expression of 7B2 mRNA and protein. HDMECs (2 3 106 cells) were left untreated (control) or stimulated with IL-1b (1 ng per ml). After RT-PCR of total RNA isolated 3 h after treatment, the ampli®cation product of 7B2 (445 bp) was separated on an agarose gel.

Little is known about the cell-speci®c expression and molecular regulation of POMC processing proteolytic enzymes in other skin cells. Here we have reported that PC1 mRNA is expressed in HDMECs and regulated by UV, IL-1, and a-MSH itself. These observations seem to be of particular importance as to date the expression of the POMC prohormone processing enzymes PC1 and PC2 seemed to be exclusively restricted to cells and tissues of endocrine and neuroendocrine origin (Castro et al, 1997). So far, the exact mechanism of PC regulation in HDMECs by MSH or IL-1 is unknown. Stimulation of protein kinase C and cAMP was demonstrated to regulate PC mRNA expression (Mania-Farnell and Davis, 1996). Thus, one could speculate that HDMEC a-MSH treatment resulting in MC-1R activation and subsequently increased intracellular cAMP (Scholzen et al, unpublished) in an autocrine manner may elevate PC1 mRNA levels in HDMECs via cAMP-dependent pathways. IL-1, which is known to activate protein kinase C, may upregulate PC1 in HDMECs involving protein kinase C. In addition to PC1, HDMECs were also found to express mRNA of the PC2 binding protein 7B2. This is of particular importance as 7B2 was demonstrated to be a critical cofactor for the autocatalytic conversion of the zymogen proPC2 that generates the full biologically active enzyme PC2 (Benjannet et al, 1995, 1998). Interestingly, according to our data HDMECs do not express PC2 mRNA that is necessary to generate a-MSH in melanotrophic pituitary cells. PC2-speci®c PCR products were detected, however, when cDNA prepared from pituitary tissue was subjected to PCR ampli®cation, which served as a positive control (data not shown). Nevertheless, a-MSH could be detected in unstimulated HDMECs by immunohistochemistry (data not shown) and we have demonstrated the presence of a-MSH in supernatants of unstimulated and stimulated HDMECs. This might in part be explained by the heterogenicity of PC2 mRNA splicing resulting in a much longer PC2 transcript in the skin compared with other tissues including pituitary (Seidah et al, 1999). Thus, we cannot exclude that the PC2-speci®c primers used in our studies were unable to detect this longer transcript, as these primers were deducted from a human pituitary PC2 mRNA sequence. Nevertheless, it is also possible that a different, not yet identi®ed, endopeptidase accounts for POMC prohormone processing in order to generate a-MSH in certain skin cells. For example, the expression of the zinc metalloprotease neutral endopeptidase (NEP; EC 3.4.24.11; also known as CD10 or enkephalinase) has been demonstrated in human cells including HDMECs (Olerud et al, 1999). NEP has been shown to process POMC to a-MSH and other peptides in invertebrates and higher vertebrates including man (Smith et al, 1992; Salzet et al, 1997). Taken together, our data indicate that UV light is capable of simultaneously upregulating the expression not only of POMC in HDMECs but also of PC1, one of the major proteolytic enzymes required to process the POMC prohormone presumably involving IL-1 as a key mediator. This could implicate that under certain experimental conditions the expression of neuropeptide convertases and their substrates might be synergistically regulated by similar mediators on the transcriptional or translational level.

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In a recent study we have demonstrated that a-MSH is capable of signi®cantly impairing lipopolysaccharide (LPS)- or TNF-ainduced MC-1R-mediated endothelial cell VCAM-1 and ESelectin mRNA and protein expression in vitro. This was mediated by inhibition of TNF-a- or LPS-induced NF-kB activation and nuclear translocation (Kalden et al, 1999). Moreover, in a mouse model for the local Shwartzman reaction systemic a-MSH treatment signi®cantly reduced the sustained expression of Eselectin and VCAM-1 as well as the development of lesions that normally characterizes this LPS-induced leukocytoclastic vasculitis (Sunderkotter et al, 1999). Thus, endothelial-cell-produced aMSH may locally serve to dampen an in¯ammatory response. The simultaneous expression of POMC peptides and their corresponding receptors may be important not only for the autocrine regulation of dermal endothelial cell functions. One could speculate that a-MSH and ACTH, if released by HDMECs into the vascular lumen in vivo, might locally or systemically achieve concentrations suf®ciently high to alter functions of circulating or adherent leukocytes. Due to their multiple anti-in¯ammatory and immunosuppressive effects endothelial-cell-derived POMC peptides may signi®cantly contribute to UV-mediated in¯ammation and immunomodulation. The grants NHI HD33024, NHI AI41493, and a VA Merit Review Award to J. Ansel, NHI R03AR44969 to C. Armstrong, and SFB293/B2 and So 87/ E1 from the Deutsche Forschungsgemeinschaft to T. Luger supported this work. T. Scholzen was supported by a fellowship grant from the Deutsche Forschungsgemeinschaft (Scho 629/1±1). The authors wish to thank M.L. HuÈlsmann for her expert secretarial assistance.

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