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Endocrinology Vol. 148, No. 6 2793-2805
Copyright © 2007 by The Endocrine Society

Terminating the Stress: Peripheral Peptidolysis of Proopiomelanocortin-Derived Regulatory Hormones by the Dermal Microvascular Endothelial Cell Extracellular Peptidases Neprilysin and Angiotensin-Converting Enzyme

Thomas E. Scholzen1, Simone König1, Michaela Fastrich, Markus Böhm and Thomas A. Luger

Ludwig-Boltzmann Institute of Cell Biology and Immunobiology of the Skin (T.E.S., M.F., M.B., T.A.L.) and Department of Dermatology (M.B., T.A.L.), Integrated Functional Genomics (S.K.), Interdisciplinary Center for Clinical Research, Medical Faculty, University of Münster, 48149 Münster, Germany

Address all correspondence and requests for reprints to: Thomas E. Scholzen, Ph.D., Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, Department of Dermatology, University of Münster, Von-Esmarch-Strasse 58, 48149 Münster, Germany. E-mail: thoscho{at}uni-muenster.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The skin including the microvascular endothelium is an established peripheral source and target of the immunomodulatory proopiomelanocortin (POMC) peptides ACTH and {alpha}-MSH. Whereas intracellular POMC peptide generation is well characterized, less is known on their extracellular processing in peripheral tissues by the neuropeptide-specific zinc metalloproteases neprilysin (NEP) and angiotensin-converting enzyme (ACE). This may locally control POMC peptide bioavailability and activation of ACTH/{alpha}-MSH-specific melanocortin receptors (MCs). In a cell-free system, endothelial cell (EC) membranes prepared from ACEhigh/NEPlow-expressing primary human dermal microvascular ECs and the ACElow/NEPhigh expressing EC line HMEC-1 degraded ACTH1–39 over time, resulting in temporary increased {alpha}-MSH immunoreactivity. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy peptide mapping and electrospray ionization-mass spectroscopy sequencing identified several stable fragments generated from ACTH1–39, ACTH1–24, and {alpha}-MSH by EC membranes or recombinant NEP and ACE. Whereas some fragments could be assigned to a cell-specific NEP or ACE activity, other degradation products require additional enzyme activity. Pharmacological NEP inhibition enhanced the ACTH and {alpha}-MSH-mediated activation of EC ectopically expressing MC1. Likewise, selected peptides such as {alpha}-MSH2–12 generated from ACTH1–39 and {alpha}-MSH by recombinant NEP displayed equipotent MC1-activating properties in vitro and antiinflammatory activity in murine allergic contact dermatitis in vivo as compared with the parental peptides. Thus, NEP and ACE significantly contribute to the EC processing of stress hormones (ACTH) and antiinflammatory peptides ({alpha}-MSH), which modulates MC1 activation but does not completely inactivate the peptide ligand. Because NEP and ACE are regulated by inflammatory mediators and UV light, this may be important for ACTH/MSH-modulated skin inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GENERATION OF regulatory oligopeptides from larger precursor molecules as well as the proteolytic degradation of peptides by specific proteases plays a pivotal role for controlling the bioavailability of peptide mediators in tissue and serum. For instance, endoproteolytic prohormone convertases (PCs) are important for the generation and secretory release of bioactive hormones and neuropeptides such as insulin, somatostatin (1), pituitary adenylate cyclase activating polypeptide (2), or the neuroendocrine proopiomelanocortin (POMC) peptides ACTH and {alpha}-MSH (3, 4). The latter are important mediators for the central as well as the peripheral response to invasive and noninvasive exogenous stress. As such, they are part of an intrinsic cutaneous hypothalamus-pituitary-adrenal axis (5). PCs also participate in the activation of extracellular matrix proteins, enzymes, and the generation of differentiation and growth factors (6). Likewise, enzymes such as mast cell tryptase or chymase are capable of cleaving and inactivating peptides including calcitonin gene-related peptide, vasoactive intestinal peptide, or the tachykinins substance P (SP) and neurokinin A in the extracellular space with high efficacy (7). More recently, the two mechanistically related zinc metalloproteases, neprilysin (NEP; EC 3.4.24.11; CD10) (8) and angiotensin converting enzyme (ACE; EC 3.4.15.1; CD143) (9), have been implicated in limiting the bioavailability of peptide mediators and growth factors to corresponding high-affinity receptors expressed on cellular targets. NEP (94 kDa) is a cell surface zinc metalloproteinase belonging to the thermolysin-like family of peptidases that degrades substrates by endoproteolytic cleavage N terminal of hydrophobic amino acid residues (for review see Ref. 8). The expression of NEP is highly restricted in hematopoietic cells but abundant in the kidney, small intestine, brain, airways, and the vascular endothelium (7). In the skin, NEP is expressed in vascular endothelial cells (ECs), dermal fibroblasts, keratinocytes, and skin appendages (10, 11, 12, 13). Initially, enkephalins and tachykinins were identified as substrates for NEP, pointing toward a role for this enzyme in turning off neuronal signals (14). Subsequently additional substrates such as bradykinin (BK) or angiotensin (Ang) I, and most recently ß-amyloid peptide, a suspected initiator of Alzheimer’s disease, have been discovered (8).

In mammalian cells, two distinct ACE isoenzymes have been identified. Somatic ACE (150–180 kDa) is a type I C terminally membrane-anchored glycoprotein consisting of two highly homologous extracellular domains. Somatic ACE activity is associated with the vascular endothelium or the renal epithelium, and a soluble ACE circulates in the plasma (9). ACE exerts dipeptidyl carboxypeptidase or endopeptidase activity in a Zn2+, chloride and substrate-dependent manner. Importantly, tissue ACE regulates the local renin-angiotensin system by cleaving Ang I into the vasoconstricting, aldosterone-releasing, blood pressure-increasing, and proinflammatory peptide Ang II. ACE also degrades SP and inactivates the vasodilating and SP-inducing oligopeptide BK. As such, somatic ACE is essential for blood pressure regulation and the control of electrolyte homeostasis. In addition, a testicular ACE involved in male fertility exists, which contains a single catalytic site identical with the C-terminal domain of somatic ACE (15).

Previous studies suggest that thermolysin-like peptidases participate in the processing and/or degradation of POMC peptides (reviewed in Ref. 16). This may control the bioavailability of ACTH and {alpha}-MSH (16), which are high-affinity ligands of specific G protein-coupled melanocortin 1 receptors (MC1) (17). Leakage of larger POMC fragments from anterior pituitary cells into the circulation has been reported (18), and ACTH and {alpha}-MSH are potentially released from extrapituitary cells including dermal microvascular ECs (19). Likewise, in human and murine skin, ACTH/MSH-related peptides of variable length have been described (20, 21, 22). The purpose of this study was to determine the capability of the EC-derived peptidases NEP and ACE to process POMC peptides by using a bioanalytical approach and to address potential functional consequences with respect to MC1 activation in vitro and murine allergic inflammation in vivo. As outlined below, NEP and ACE are fundamentally involved in processing of POMC peptides by EC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Primary human dermal microvascular ECs (HDMECs) were obtained from PromoCell (Heidelberg, Germany). HDMECs and the cell line HMEC-1 were grown in a supplemented microvascular EC basal media (EBM-MV kit system; PromoCell, as supplied by the manufacturer) in a humidified atmosphere at 37 C and 5% CO2. Experiments were conducted with cells in passages 3–6. HDMEC cultures were characterized by their typical cobblestone morphology using light microscopy and by analysis for their capacity to express factor VIII-like antigen or CD31 (platelet endothelial cell adhesion molecule-1). Before stimulation, HDMECs or HMEC-1 were plated at a density of 25,000 cells/cm2. In some experiments (assessment of intracellular cAMP and luciferase activity), cells were deprived from growth factors and fetal bovine serum by culturing in stimulation medium for 24 h before stimulation with POMC peptides (EBM-MV, no supplements except 0.5% fetal bovine serum and antibiotics).

Flow cytometry
For the analysis of HDMECs or HMEC-1 cell surface expression, detached cells were stained with a mouse antihuman CD10 monoclonal antibody conjugated to fluorescein isothiocyanate (clone B-E3; Acris GmbH, Hiddenhausen, Germany). For the detection of ACE, an antihuman CD143 mAb (clone 9B9; Acris) followed by fluorescein isothiocyanate (FITC)-conjugated antimouse IgG antibody (Sigma-Aldrich, Taufkirchen, Germany) was used. Flow cytometry was performed on a FACSCalibur cytometer using CellQuest Pro Software (BD Biosciences, Heidelberg, Germany).

Membrane preparation
For preparation of crude cell membranes, HMEC-1 or HDMECs were grown to subconfluency (80–90%), washed with PBS, and detached with Accutase (PAA Laboratories, Pasching, Austria). Cells collected by centrifugation (300 x g) were washed with PBS and resuspended in 0.05 M Tris/HCl (pH 7.4). Subsequently cells were lysed by sonication (10 x 5 sec on ice) and subjected to 100,000 x g centrifugation (30 min, 4 C). Membrane pellets were washed twice with ice-cold 0.05 M Tris-HCl (pH 7.4), resuspended in 0.05 M Tris-HCl (pH 7.4), and the protein content was adjusted to 1 mg/ml.

Determination of NEP and ACE enzymatic activity
NEP activity of cells or cell membranes was determined using the artificial substrate glutaryl-Ala-Ala-Phe-4-methoxy-ß-naphtylamide that is converted to the fluorescent product 4-methoxy-2-naphtylamin (MNA) in the presence of aminopeptidase M as previously described (23). Fluorescence of MNA was detected at 425 nm emission wavelength in a Fluoromax-2 fluorometer with Datamax 2.2 software (Instruments S.A. Inc., Edison, NJ) and an integrated GRAMS/386 data processing and database software (Galactic Industries Corp., Salem, NH) using an excitation wavelength set to 340 nm. NEP enzyme activity was calculated from a standard curve of 0–3000 pmol MNA, which was linear in the range of 0–3000 pmol MNA and expressed in microunits with 1 µU = 1 pmol MNA per hour per microgram protein. ACE activity was determined using a protocol modified from one previously published (24). Accordingly, plasma or cell lysates were mixed in a 1:8 sample to reagent volume ratio with a solution containing 1 mM of the substrate N-(3-[2-furyl]acryloyl)-Phe-Gly-Gly and 300 mM NaCl in 80 mM boric acid (pH 8.2 at 37 C). After incubation (5 min/37 C), the reaction mixture was transferred to a semimicro glass cuvette, and the absorbance change at 345 nm was recorded after a lag phase of 2 min for 5 min every 30 sec with a Beckman spectrophotometer. ACE activity was determined from a known standard containing human recombinant ACE (Sigma Diagnostics).

Determination of ACTH and {alpha}-MSH
HDMECs and HMEC-1 cells or cell membranes, respectively, were incubated with ACTH1–39 for various time points. Cell or membrane supernatants were harvested and ACTH and {alpha}-MSH IR was determined using commercially available RIAs (EuroDiagnostica, Malmö, Sweden) as described (19).

POMC peptide processing by EC membranes or recombinant proteases
ACTH/MSH-related peptides were obtained from Bachem AG (Bubendorf, Switzerland), except {alpha}-MSH2–12, which was synthesized by IRIS Biotech GmbH (Marktredwitz, Germany). Peptide processing was performed as described (25). Briefly, peptides were incubated with defined amounts of HDMECs or HMEC-1 membrane protein in 0.05 M Tris-HCl (pH 7.4) at 37 C for various time points (1–240 min). Specific degradation by NEP or ACE was determined by the addition of NEP and ACE inhibitors [phosphoramidon (PA), thiorphan (TP), and captopril, respectively; Sigma-Aldrich]. To evaluate influences of other proteases, incubations were performed in the presence of 10 mM phenylmethylsulfonylfluoride (serine proteases), 1 mM pepstatin A (aspartic proteases), 1 mM {alpha}-iodoacetamide (cystein proteases), 10 mM of the pan-metalloprotease inhibitor 1,10 phenathroline (all from Sigma-Aldrich), or 10 mM Na-EDTA in the presence or absence of divalent cations such as Ca2+ (15 mM CaCl2) or Zn2+ (10 µM ZnCl2). The pH dependency of peptide fragmentation was analyzed in a 100 mM sodium acetate buffer at pH 5.0. For incubation with rhuNEP (generously provided by Nigel W. Bunnett, University of California, San Francisco, San Francisco, CA), peptides were incubated for 30 and 120 min at 37 C with 0.1–10 pmol (~0.01–1.0 µg) rhuNEP in 0.05 M Tris-HCl (pH 7.4) in the presence or absence of NEP and ACE inhibitors or 10 µM ZnCl2, respectively. Incubations of {alpha}-MSH and ACTH1–39 with 1.7 and 0.17 pmol rhuACE (0.2 and 0.02 µG protein, respectively; R&D Systems GmbH, Wiesbaden, Germany) were performed at 37 C in 0.1 M Tris/HCl (pH 8.3), 0.3 M NaCl, and 10 µM ZnCl2. The reactions were terminated by the addition of 10 µM NEP or ACE inhibitors, respectively.

Peptide purification and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)
Soluble peptides were separated from cell membranes or recombinant proteins by ultra filtration at 4 C through Amicon Microcon centrifugal filter devices (cut-off size: 10 kDa; Millipore GmbH, Schwalbach, Germany), lyophilized and desalted using C18 ZipTips (Millipore) as described (25). Peptide maps were generated with a TofSpec-2E instrument (Micromass/Waters, Manchester, UK) in reflection mode using {alpha}-cyano-4-hydroxycinnamic acid as matrix and 1 µl of analyte. Products were identified using a manual nanospray MS/MS on a Esquire3000 iontrap instrument (Bruker Daltonik GmbH, Bremen, Germany).

Transfection
ECs and cells of the embryonic kidney cell line HEK293 were transfected by electroporation (modified from Ref. 26) with a pcDNA3.1+ expression vector-based cDNA expression construct (University of Missouri-Rolla cDNA Resource Center, Rolla, MO) containing either the full-length human MC1 cDNA (GenBank accession no. NM_002386) or the human MC1 cDNA with a 3x N-terminal hemagglutinin (HA) tag (HA-MC1). In some experiments, ECs were transfected with a PathDetect luciferase cis-reporter plasmid (pCRE-Luc; Stratagene, La Jolla, USA) that contains the luciferase gene under control of a basic promoter (TATA box) and an inducible 4 x cAMP responsive element (CRE) consensus sequence (AGCCTGACGTCAGAG)4. Semiconfluent cells were harvested, washed with PBS, and resuspended in 0.6 ml cold transfection buffer [137 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 6 mM glucose, 0.1 mM ß-mercaptoethanol, and 1.0–2% dimethyl sulfoxide in 20 mM HEPES (pH 7.0)] at a density of 1 x 107 cells/ml. Cells were transfected with 10–20 µg DNA by a single exponential decay pulse (250 V/cm, 950 µF, 20 msec) in 4-mm electroporation cuvettes using in a GenePulser Xcell system (Bio-Rad, Munich, Germany). Cells transfected with an empty vector or cells subjected to the transfection procedure only served as transfection controls. In some cases, transfection efficacy was estimated by parallel cell transfections with a pcDNA3.1+ enhanced green fluorescence protein expression vector and flow cytometry analysis. Expression of HA-MC1 was monitored by flow cytometry using a monoclonal rat-anti-HA Ab (clone 3F10; Roche Diagnostics, Mannheim, Germany) followed by a FITC-conjugated polyclonal antirat antibody (BD Biosciences).

Determination of cAMP and luciferase activity
Subconfluent cells (HMEC-1, HDMECs, HEK293) in 24-well plates were deprived from growth supplements/serum for 24 h before stimulation. Cells preincubated for 30 min with 50 µM 1-isobutyryl-3-methyl-xanthine (IBMX; Sigma-Aldrich) were stimulated with POMC peptides or forskolin (10 µM; Calbiochem) in the presence of IBMX for 15 min, and the intracellular cAMP accumulation was determined by enzyme immunoassay (GE Healthcare Life Sciences, Freiburg, Germany). To assess luciferase activity, pCRE-Luc transfected ECs were washed with PBS, lysed, and assayed using reporter lysis buffer and luciferase assay buffer (Promega Corp., Madison, WI). Samples were read for 10 sec in a Gen-Probe single tube luminometer (MGM Instruments Inc., Hamden CT).

Sucrose density gradient centrifugation and Western blotting
Untransfected or HA-MC1-transfected ECs were subjected to sucrose gradient density centrifugation as described with modifications (27). Briefly, 2–3 x 107 ECs were detached with trypsin and lysed in 1 ml lysis buffer [25 mM Tris-HCl (pH 6.8), 150 mM NaCl, 1% Triton X-100, 1x Complete (Roche Diagnostica)] for 15 min on ice. The lysate was sheered by 15 strokes through a 30-gauge needle, adjusted to 40% sucrose by the addition of an equal volume of 80% sucrose in the above buffer without detergent and carefully overlaid with a discontinuous gradient prepared of 4 ml 30% sucrose and 4 ml 5% sucrose, respectively. After centrifugation for 18 h in a SW41 rotor at 200,000 x g (35,000 rpm) in a Beckman Optima L-70K ultracentrifuge (Beckman Coulter GmbH, Krefeld, Germany), 1-ml fractions were harvested from the top. Proteins were precipitated by the addition of 50% trichloroacetic acid and resuspended in denaturing Laemmli buffer. Equal amounts of protein were separated by 8–12% SDS-PAGE and electroblotted on nitrocellulose membrane using standard procedures. Membranes were probed with the following primary antibodies (Ab): a mouse antihuman CD10 (1:100; clone 56C6; AbD Serotec GmbH, Düsseldorf, Germany), a goat polyclonal anti-G{alpha} q (1:500, Santa Cruz Biotechnologies Inc., Santa Cruz, CA), a polyclonal rabbit anticaveolin-1 Ab (1:5000; BD Biosciences), an anti-HA Ab (Sigma-Aldrich), or a rabbit polyclonal anti-MC1 Ab (28). Secondary antibodies were conjugated to horseradish peroxidase (Amersham Biosciences Europe GmbH, Freiburg, Germany). Bands were visualized by applying a chemiluminescence detection system (SuperSignal; Pierce Biotechnology, Rockford, IL).

Animals, induction of allergic contact dermatitis (ACD), and application of peptides
C57BL/6J mice were bred in a special pathogen-free animal facility (Department of Dermatology, University of Münster) or obtained from Harlan Winkelmann (Borchen, Germany). Mice were housed in a barrier facility with free access to water and food in compliance with federal, state, local, and institutional regulations. Males and females were studied at 8–14 wk with five to seven mice per experimental condition. ACD responses to 2,4-dinitro-1-fluorobenzene (DNFB; Sigma-Aldrich) were induced and elicited essentially as previously described (29). To determine POMC peptide effects on ACD elicitation, 10 µl of peptides solubilized in vehicle (2% dimethylsulfoxide, 48% ethanol, 50% H2O) were applied on the ventral surface of the challenged ear 60 min before and 15 min after antigen challenge. Sensitized mice treated with vehicle before challenge served as control.

Statistical analysis
Results are expressed as arithmetic mean ± SEM. The unpaired t test was used to calculate the statistical significance. Differences between multiple groups were examined using an ANOVA (Bonferroni t test). Mean differences with P < 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of endothelial NEP and ACE
To analyze the relative expression of neprilysin and ACE in ECs, untreated primary dermal microvascular ECs or HMEC-1 were stained with FITC-conjugated antibodies against human NEP or ACE, respectively. HDMECs express 40–55% ACE but only up to 15% NEP, whereas HMEC-1 display a reciprocal NEP and ACE expression profile corresponding to 40–50% NEP and up to 10% ACE, respectively (Fig. 1Go).


Figure 1
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  		FIG. 1. NEP and ACE expression in microvascular ECs. Primary HDMEC (top) or cells of the EC line HMEC-1 (bottom) were stained with FITC-conjugated monoclonal Abs against human ACE (open histogram, bold line) or NEP (shaded histogram) and subjected to flow cytometry.

 
Degradation of ACTH1–39 by ECs
To determine, whether EC are capable of degrading or processing POMC peptides, cell membranes prepared from the EC cell line HMEC-1 were incubated with ACTH1–39. When the ACTH content in membrane supernatants was monitored over time by using an ACTH-specific RIA, ACTH1–39 continuously decreased to the level of controls after 4 h (Fig. 2AGo). Interestingly, in the same supernatants, {alpha}-MSH immunoreactivity (IR) temporarily increased peaking at 60 min after the addition of ACTH (Fig. 2BGo). This generation of {alpha}-MSH IR could be prevented by the addition of an NEP inhibitor (PA), indicating that an NEP-like protease participates in this cleavage (Fig. 2CGo).


Figure 2
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  		FIG. 2. Processing and generation of ACTH and {alpha}-MSH by EC membranes. ACTH1–39 was incubated with 100 µg human microvascular endothelial cell line-1 membranes and the IR for ACTH (A) and {alpha}-MSH (B and C) in the supernatants was monitored by RIA at the time points indicated. Note that ACTH IR decreases over time (A), whereas {alpha}-MSH IR is temporarily increased after 60 min (B) in an NEP-dependent manner (C). Values are expressed as mean ± SEM (n = 3 independent experiments); *, P < 0.05 (vs. control); **, P < 0.01 (vs. control).

 
Fragmentation of POMC peptides by EC cell membranes
Next, the fate of ACTH1–39 after incubation with HDMECs (Fig. 3Go) and HMEC-1 membranes (Fig. 4Go) was investigated in detail. After incubation with HDMEC membranes, MALDI-TOF analysis revealed fragmentation of ACTH1–39 to a number of larger fragments that included ACTH2–17, corticotropin-like intermediate peptide (ACTH18–39), ACTH17–39, ACTH16–39, and ACTH15–39. Likewise, a number of smaller fragments 5–11 amino acids in length derived from the N terminus of the molecule, such as ACTH5–10, ACTH5–12, ACTH5–13, ACTH5–14, ACTH5–15, and ACTH5–16 could also be detected. Inhibition of NEP by PA/TP predominantly inhibited the generation of N-terminal ACTH fragments (Fig. 3BGo), whereas ACTH18–39, ACTH2–17, ACTH17–39, ACTH16–39, and ACTH15–39 were still detectable. In contrast, after inhibition of ACE by captopril peptides with mass/charge (m/z) greater than 2100 were no longer detectable (Fig. 3CGo). When ACTH1–39 was incubated with recombinant human ACE (rhuACE), peptides such as ACTH5–14, ACTH18–39, ACTH17–39, or ACTH16–39 were identical with those detected after incubation with HDMEC membranes in the absence of enzyme inhibitors. Therefore, ACE, which is abundantly expressed by HDMECs, may to a major degree be responsible for the generation of these peptides by HDMECs membranes (Fig. 3DGo).


Figure 3
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  		FIG. 3. Fragmentation of ACTH by HDMEC membranes and recombinant ACE. ACTH1–39 was incubated with HDMEC membranes for 120 min at 37 C in the absence (A) or presence of 1 µM PA/TP each (B) or captopril (C) or were incubated with rhuACE (D). The resulting fragments were purified, desalted, and subjected to MALDI-TOF analysis. Notably, some but not all fragments generated by rhuACE processing were identical with those generated HDMEC membranes.

 

Figure 4
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  		FIG. 4. Fragmentation of ACTH and {alpha}-MSH by HMEC-1 membranes. ACTH1–39 (A and C) or {alpha}-MSH (B and D) was incubated for 60 min at 37 C with cell membranes from HMEC-1 without enzyme inhibition (A and B) or in the presence of the NEP inhibitors PA/TP (1 µM) each (C and D). The resulting fragments were purified, desalted, and subjected to MALDI-TOF analysis (A–D). Note that the fragmentation profile of ACTH is changed by the addition of the NEP inhibitors (C), and this addition also reduces the intensities of some of the detected {alpha}-MSH fragments or removes them (D).

 
When ACTH1–39 (Fig. 4AGo) and also ACTH1–24 (data not shown) were incubated with HMEC-1 membranes, ACTH2–17 as well as ACTH5–12 was identified as the predominant peptide fragment. However, depending on the time point of analysis, membrane supernatants also contained other stable peptides such as ACTH4–9, ACTH5–10, ACTH3–12, ACTH4–12, ACTH5–14, or ACTH3–14. In contrast, after incubation of ACTH1–39 with HMEC-1 membranes in the presence of NEP inhibitors, a significant change in the cleavage pattern of ACTH was observed (Fig. 4CGo). Rather than before V13, cleavage now occurred predominantly before K15, and the intensities of both ACTH5–12 and ACTH2–17 were clearly diminished. When HMEC-1 membranes were incubated with {alpha}-MSH, this peptide was proteolytically processed to fragments depicted in Fig. 4BGo. After the addition of NEP inhibitors, only {alpha}-MSH1–12, {alpha}-MSH4–13, and {alpha}-MSH5–13 remained detectable with lower intensities, which could be further reduced or completely removed, respectively, by lowering the amount of membrane protein used for the incubation (see Fig. 4DGo and Table 3Go).


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  		TABLE 3. Qualitative intensity values of {alpha}-MSH fragmentation after digestions with rhuNEP (1, 0.1 µg) or human microvascular membranes (100, 10 µg)

 
Fragmentation of POMC peptides by recombinant enzymes
Next, ACTH1–39 and {alpha}-MSH were incubated with recombinant human NEP (rhuNEP) or rhuACE to identify POMC cleavage products that were specifically generated by these enzymes. Stable peptides identified after incubation of ACTH with rhuNEP were predominantly cleaved before hydrophobic amino acids (V, Y, F) (Table 1Go). Likewise, when {alpha}-MSH was incubated with rhuNEP, a characteristic cleavage pattern could be observed (Fig. 5AGo and Table 2Go), which depended on the amount of protease used (Table 3Go). Notably, some cleavage products were identical with those obtained with HMEC-1 membranes (Fig. 4Go, A and B). This {alpha}-MSH proteolysis could be dramatically reduced by the addition of NEP inhibitors (PA/TP; Fig. 5CGo), whereas the addition of the ACE inhibitor captopril remained virtually ineffective (Fig. 5EGo). These data clearly suggest that NEP to a greater extent participates in HMEC-1 {alpha}-MSH processing. In addition to the fragments indicated in Fig. 5AGo, NEP to a minor extent and after prolonged incubation (120 min) also generated {alpha}-MSH8–13, {alpha}-MSH7–13, {alpha}-MSH6–13, {alpha}-MSH5–13, {alpha}-MSH7–12, and {alpha}-MSH2–8, respectively. Likewise, rhuACE cleaved {alpha}-MSH after H6, F7, and G10 (Fig. 5BGo). In the presence of the ACE inhibitor captopril, ACE degraded {alpha}-MSH from the N terminus (Fig. 5DGo). All stable fragments obtained were verified by electrospray ionization (ESI)-MS/MS as exemplified for MSH2–12 (Fig. 5FGo). Selected peptides such as {alpha}-MSH2–12, which was generated from ACTH1–39, ACTH1–24, and {alpha}-MSH by rhuNEP and also in small amounts by EC membranes, was used in functional studies as outlined below.


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  		TABLE 1. Cleavage of ACTH1–39 by rhuNEP after 30 min

 

Figure 5
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  		FIG. 5. Fragmentation of {alpha}-MSH by recombinant (rec) NEP and ACE. {alpha}-MSH was incubated for 60 min at 37 C with 1 pmol (about 1 µg) of rhuNEP or rhuACE, respectively, without enzyme inhibition (A and B) or in the presence of the NEP inhibitors PA/TP (1 µM) each (E) or the ACE inhibitor captopril (C and D). The resulting fragments were purified, desalted, and subjected to MALDI-TOF analysis (A–D). Note that the addition of the NEP inhibitors (C) but not the ACE inhibitor (E) effectively prevents {alpha}-MSH degradation.

 

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  		TABLE 2. Cleavage of {alpha}-MSH by rhuNEP after 30 and 120 min

 
Fragmentation of POMC peptides by EC membranes mostly generated expected but also somewhat unexpected fragments that could not be attributed to exclusive NEP or ACE activity. When ACTH and {alpha}-MSH were incubated with HMEC-1 membranes (pH 5.0 instead of pH 7.5), a predominant cleavage of ACTH into ACTH4–10, ACTH1–10, and ACTH4–14 and {alpha}-MSH into {alpha}-MSH1–9, and {alpha}-MSH2–11, respectively, was observed (data not shown). At this pH, inhibition of NEP or ACE remained virtually ineffective, suggesting that products were not generated by these peptidases. In addition, incubation of ACTH with HMEC-1 membranes in the presence of serine protease inhibitors (phenylmethylsulfonylfluoride) resulted in a partially reduced generation of ACTH2–17, suggesting that serine proteases may participate in the generation of this peptide. Likewise, the addition of EDTA resulted in an overall reduction of peptide fragments, particularly of ACTH5–10, ACTH5–12, or ACTH5–14, indicative of a pivotal role of divalent cations in the processing of ACTH (data not shown).

Functional aspects of POMC peptide fragmentation on MC signaling
To analyze the functional consequences of POMC peptides processing by NEP or ACE for the activation of EC MC1 receptors, HMEC-1 cells were stimulated with various concentrations of {alpha}-MSH or ACTH1–39. After 15 min, a dose-dependent induction of cAMP was observed (Fig. 6Go, A and B). Notably, when HMEC-1 cells were transfected with a HA-tagged-MC1 expression vector, the cAMP induction by POMC peptides but not that induced by the adenylyl cyclase-stimulating agent forskolin (FSK) was significantly increased in comparison with unstimulated controls, and cells not transfected with HA-MC1 (Fig. 6Go, A and B). To determine, whether proteolytic processing of {alpha}-MSH or ACTH1–39 potentially modulates MC1-signaling, cells were incubated with synthetic {alpha}-MSH2–12 as a model peptide, which was identified by MS/MS in supernatants after incubation of {alpha}-MSH or ACTH1–39 with both rhuNEP and NEP-expressing HMEC-1 cells (Tables 1–3GoGoGo and Fig. 4AGo). In MC1-transfected HMEC-1 (Fig. 6CGo) or HEK293 cells (Fig. 6DGo) {alpha}-MSH2–12, but not the C-terminal tripeptide KPV ({alpha}-MSH11–13) is still capable of inducing cAMP amounts comparable with native {alpha}-MSH or full-lengthACTH1–39 (Fig. 6Go, C and D). Thus, a C- and N-terminal truncation of {alpha}-MSH does not impair MC1 signaling. Stimulation of MC1-transfected HMEC-1 with POMC peptides in the presence of PA also significantly increased the intracellular content of cAMP in comparison with cells not treated with this NEP inhibitor (Fig. 6EGo), indicating that NEP inhibition augments functional MC1 activity. Noteworthy, NEP inhibition also increased activity of MSH2–12 on the MC1 suggesting that this peptide may subject to further degradation by PA-inhibited enzymes (Fig. 6FGo). Moreover, ACTH1–39, {alpha}-MSH2–12 and FSK induced luciferase activity in HMEC-1 cells transfected with the luciferase cis-reporter plasmid pCRE-Luc (Fig. 6FGo). Thus, cAMP accumulation induced by POMC peptides after MC1 activation results in downstream activation of CRE-regulated gene expression.


Figure 6
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  		FIG. 6. Modulation of MC1 signaling in HMEC-1 and HEK293 cells. HMEC-1 were transfected with a HA-tagged expression construct containing the full-length cDNA of human MC1 (HA-MC1). Transfected and untransfected control cells pretreated with IBMX were stimulated for 15 min with various concentrations of {alpha}-MSH (A), ACTH1–39 (B), or the {alpha}-MSH peptides {alpha}-MSH2–12 and {alpha}-MSH11–13 (C), and the intracellular cAMP content was determined by enzyme immunoassay. Treatment with 10 µM FSK served as control in these experiments. HEK293 were HA-MC1-transfected (D) and stimulated with 10–7 M of the indicated peptides. ACTH1–39, {alpha}-MSH, {alpha}-MSH2–12, but not {alpha}-MSH11–13 elicited a cAMP response in transfected but not untransfected HEK293 cells. HA-MC1-transfected HMEC-1 cells were treated with 10–7 M ACTH1–39, {alpha}-MSH, or {alpha}-MSH2–12 in the presence of 1 µM of the NEP inhibitor PA (E). HMEC-1 cells transfected with the luciferase reporter plasmid pCRE-Luc were stimulated with ACTH1–39, {alpha}-MSH2–12, or FSK for 15 min, and luciferase activity was determined after 4 h. (F). The cAMP contents (A–D and F) or the luciferase activity (F) are expressed in percent as mean ± SEM with unstimulated controls set to 100% (n = 3 independent experiments). *, P < 0.05 vs. control; **, P < 0.01 vs. control; ***, P < 0.001 vs. control (A–D and F); #, P < 0.05 vs. cells not treated with the NEP inhibitor (E).

 
Colocalization of components that modulate MC1 signaling in membrane rafts
Signaling and endocytosis of G protein-coupled receptor (GPCR) frequently requires association of receptor components in membrane microdomains (rafts) enriched for cholesterol and caveolin (30). In HA-MC1 transfected HMEC-1, treatment with the cholesterol-binding and -depleting agent filipin impaired the capability of {alpha}-MSH and the proteolysis-resistant analog [Nle4, D-Phe7]-{alpha}-MSH to induce cAMP (Fig. 7AGo), suggesting that intact, cholesterol-rich membrane microdomains are required for MC1 signaling. Next, HA-MC1-transfected HMEC-1 cells were lysed in the cold in the presence of Triton X-100 and subjected to sucrose gradient fractionation. Interestingly, after SDS-PAGE and Western blotting, MC1 IR could be detected in low-density gradient fractions positive for caveolin-1 (Fig. 7BGo) and other membrane markers such as G{alpha} q (data not shown). We also detected NEP IR in these low-density but also in high-density fractions, favoring the idea that NEP and MC1 may be colocalized in distinct membrane rafts.


Figure 7
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  		FIG. 7. Impairment of MC1 signaling by lipid raft disruption and localization of NEP and MC1 in raft-enriched membrane fractions. HA-MC1-transfected HMEC-1 cells were treated for 60 min with filipin and subjected to simulation with {alpha}-MSH and [Nle4, D-Phe7]-{alpha}-MSH. Note that filipin treatment reduces cAMP response to both peptides, indicating that MC1 signaling may require intact cholesterol-rich membrane microdomains (A). Cell lysates were prepared from by HA-MC1 transfected HMEC-1 and subjected to differential centrifugation and sucrose gradient centrifugation. After SDS PAGE and Western blotting using antibodies against NEP/CD10, caveolin-1, and MC1, MC1 IR was predominantly detected in fraction 4 and 5, which also displayed IR for NEP and the raft markers caveolin-1 (B) and G{alpha} q (data not shown). Data are presented in percent as mean ± SEM with unstimulated controls set to 100% [n = 3 independent experiments and *, P < 0.05 vs. control (A)] or are representative of two independent experiments (B).

 
Effect of selected POMC peptides on ACD in mice
{alpha}-MSH is an established, highly potent antiinflammatory and immunosuppressive mediator (31). To examine, whether {alpha}-MSH2–12 in addition to its MC1-activating properties in vitro is still antiinflammatory in vivo, we analyzed its capacity to modulate murine ACD responses in comparison with ACTH and {alpha}-MSH. Accordingly, when DNFB-sensitized mice were epicutaneously treated with the respective peptides before and after antigen ear challenge, {alpha}-MSH2–12, in addition to {alpha}-MSH and/or ACTH1–39, displayed a significant anti-inflammatory activity on ACD elicitation as revealed by reduced allergic ear swelling responses in comparison with mice not treated with the peptides (Fig. 8Go). Reduced ear-swelling responses were accompanied by clearly reduced histological signs of inflammation 48 h after antigen challenge in ears treated with the peptides (data not shown). The effect of peptide application on the ear swelling without antigen challenge as well as DNFB treatment of mice not sensitized with DNFB did not exert any significant change in ear swelling (data not shown). Thus, the truncated NEP-derived fragment {alpha}-MSH2–12 retains its antiinflammatory properties in this model of skin inflammation.


Figure 8
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  		FIG. 8. Reduced ACD responses after local POMC peptide application. C57BL/6J mice were sensitized with 0.5% DNFB on the shaved abdomen at d 0 and challenged with 0.2% DNFB on the right ear at d 5 as previously described (29 70 ). ACTH1–39, {alpha}-MSH, or MSH2–12 was locally applied on the right ear 60 min before and 15 min after antigen challenge. The ACD response was determined by the degree of ear swelling of the hapten-exposed ear, compared with that of the vehicle-treated contralateral ear before DNFB challenge and at 24 h after challenge. Ear swelling values are given in micrometers as mean ± SEM (n = 7). **, P < 0.01 for peptide-treated mice vs. control mice (sensitization and challenge without peptide application).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we demonstrate that NEP and ACE, two important ectopic peptidases expressed by ECs, are highly relevant for the proteolytic processing of POMC peptides such as ACTH1–39, ACTH1–24, and {alpha}-MSH. We used membranes from HDMECs and a HDMEC-derived EC line (32) that share EC-phenotypical properties (33) but notably differ in their reciprocal expression and regulation of NEP and ACE (34, 35). A similar inversely correlated regulation of NEP and ACE expression has been observed in spontaneously hypertensive rats (36), and the opposing roles of ACE and NEP due to a competitive cleavage of vasoactive peptides such as angiotensin or bradykinin are highly relevant for cardiovascular homeostasis. Thus, the above cells constitute a suitable EC model to analyze the role of a differential peptidase expression on the processing of external mediators.

Incubation of ACTH1–39 with EC membranes clearly decreased the ACTH IR in the membrane supernatants over time, causing a temporary NEP-dependent increase in {alpha}-MSH IR. A similar processing of ACTH1–39 or a parasite-derived POMC-like hormone by NEP-expressing human granulocytes or invertebrate immunocytes, respectively, has been reported (37, 38, 39). Although this suggested a NEP-dependent generation of bioactive {alpha}-MSH, the authors did not rule out an endogenous {alpha}-MSH release in their cell system. In contrast, we excluded the possibility that EC-derived POMC peptides (19) may interfere with the analysis of POMC peptide processing by using a cell-free system. Moreover, MALDI-TOF MS and ESI-MS analysis did not reveal the presence of {alpha}-MSH (acetyl-ACTH1–13-amid) after incubation of ACTH1–39 with EC membranes or rhuNEP, thus excluding a direct ACTH-to-{alpha}-MSH conversion. In addition, we identified several larger and shorter ACTH fragments that may have caused false-positive results in the {alpha}-MSH RIA used.

The specificity of some but not all of the fragments generated from ACTH1–39 or ACTH1–24 by EC membranes could be assigned to NEP or ACE activity by switching the pH to a value suboptimal for these enzymes, applying specific enzyme inhibitors, or comparing the peptide profile with that generated by recombinant enzymes. Likewise, a peptide at m/z 1662 was routinely detected, which did not correspond to any regular proteolytic peptide but could be derived from ACTH1–14 with an intramolecular loss of water. This dehydration process originates from the sample and not from MS processes and the assignment was supported by ESI-MS/MS. However, there are a number of open questions surrounding that peptide and its purification is in progress for further clarification. Importantly, EC membranes also digested {alpha}-MSH to peptides such as MSH/ACTH4–12, MSH4–13, MSH/ACTH2–12, or MSH2–13. A preferred cleavage occurred in the N terminus of the ACTH/{alpha}-MSH molecule at positions S1-Y2, H6-F7, and K12-V13 ({alpha}-MSH, ACTH), P18-V19, or K21-V22 (ACTH), which is in good agreement with the proposed cleavage pattern for NEP N terminal of large hydrophobic amino acid residues (40). In addition, rhuNEP and particularly EC membranes also cleaved {alpha}-MSH/ACTH1–39 at somewhat unexpected sites (M4-E5, E5-H6, G9-K10, G14-K15, or R17-R18). Thus, ACE and particularly NEP specificity is not always confined to the above cleavage pattern, and with respect to NEP, variations have been described. In general, NEP has broader substrate specificity and cleaves peptides as large as 17 kDa, although true endopeptidase activity preferentially occurs within smaller linear peptides such as tachykinins, gastrin, or cholecystokinin-8 (41).

ACE has two structurally related zinc binding sites that both posses endopeptidase and dipeptidyl carboxypeptidase activity but differ with respect to pH/chloride dependency and substrate specificity (42, 43). For example, a higher selectivity of the ACE inhibitor captopril described for the N-terminal catalytic site (44) may partly explain why inhibition of ACE by captopril, rather then completely preventing ACE-dependent {alpha}-MSH cleavage, resulted in degradation of MSH from the N terminus. Interestingly, NEP inhibition of ACTH degradation by ACEhigh/NEPlow-expressing HDMECs predominantly resulted in a loss of N terminus-derived ACTH degradation products, whereas in contrast, ACE inhibition prevented the generation of larger C-terminal fragments (e.g. ACTH18–39 or ACTH15–39). Thus, NEP and ACE may competitively cleave ACTH, which is a common way of modulating substrate bioactivity. For instance, whereas ACE cleaves Ang I to the hypertensive and proinflammatory peptide Ang II (Ang1–8), NEP generates Ang1–7, which acts as endogenous inhibitor of Ang II (45). Interestingly, the presence of peptides such as {alpha}-MSH1–10 or {alpha}-MSH3–10 after ACE-mediated {alpha}-MSH cleavage suggest that the ACE carboxypeptidase activity could be of in vivo relevance for the liberation of the C-terminal tripeptide {alpha}-MSH11–13 (KPV), which is sufficient to mimic {alpha}-MSH-like antiinflammatory and immunosuppressive activities in vivo (46). In line with our findings, melanoma cell lines (47) as well as melanocytes displayed the ability to process {alpha}-MSH in a PA-sensitive way indicating a (patho)-physiological role for NEP in malignant melanoma as well as in pigmentation (48). Likewise, ACE pH-dependently metabolizes ACTH1–39 in the central nervous system, which may be particularly relevant for pituitary functions (Ref. 16 and references therein). For instance, ACTH1–39 was predominantly degraded to ACTH1–16, ACTH17–39, ACTH22–39, and ACTH3–15 by rat brain synaptic membranes at pH 8.5 (49), which resembled the ACTH1–39 peptide profile obtained from HDMECs or rhuACE in our experiments. The detected ACTH1–15 and ACTH1–14 could be derived from ACTH1–16 trimming by residual ACE carboxypeptidase activity.

MC receptors in mouse and men signal via coupling to heterotrimeric G proteins, resulting in adenylase cyclase-dependent cAMP synthesis, protein kinase A activation, and CRE-dependent gene expression (reviewed in Refs. 17 , 50). In accordance with this notion and similar observations for HDMECs (51), ACTH1–39 or {alpha}-MSH induced cAMP in NEPhigh-expressing HMEC-1 as well as luciferase activity in CRE-luciferase reporter-transfected cells. {alpha}-MSH and ACTH displayed a biphasic stimulatory effect on cAMP in the micro- as well as the lower nanomolecular range. Although there is no experimental evidence of MC receptors other than MC1 expressed on microvascular ECs (51), the recently observed homo- or heterodimerization of the MC1 (52) with a not-yet-identified partner on EC membranes may account for this effect, resulting in MC receptors with different affinities for one ligand. Importantly, POMC peptide peptidolysis was functionally relevant because the induction of cAMP was significantly augmented by the following conditions: 1) after adding NEP inhibitors, 2) by increasing the number of active binding sites after MC1 transfection, or 3) by using the proteolysis-resistant {alpha}-MSH analog Nle4-D-Phe7-{alpha}-MSH (data not shown). The latter is modified at sites particularly susceptible to NEP cleavage, indirectly confirming that NEP is a major player in {alpha}-MSH/ACTH degradation. However, we also noted that the NEP-derived ACTH/{alpha}-MSH fragment {alpha}-MSH2–12 retained MC1-activating properties in vitro and antiinflammatory bioactivity in vivo comparable with the parental peptides. In contrast, KPV did not trigger cAMP signaling, even in MC1-transfected HMEC-1 or HEK293 cells. Although KPV-induced Ca2+ signaling in HaCaT cells and keratinocytes (53), it does not bind to the MC1 or other MCx and its mode of action is still a matter of debate (50).

Several ACTH/MSH fragments identified after EC membrane or NEP/ACE processing contained the POMC peptide pharmacophore sequence HFRW (ACTH6–9), the minimum sequence required for unspecific binding to MCx. Outside this sequence, M4 and P12 are of higher importance for MC1 binding, whereas the first three N-terminal amino acids appear to be dispensable. ACTH4–10 is a weak agonist for MC1 with EC50 values in the lower micromolar range (50). In MC1-transfected HEK293 cells or human melanocytes, {alpha}-MSH and ACTH1–17 showed similar binding affinity for human MC1, whereas binding affinities for ACTH1–39, desacetyl-{alpha}-MSH, and ACTH1–10 were lower. ACTH1–17 is more potent than acetylated {alpha}-MSH in stimulating melanogenesis, indicating that this peptide is a powerful ligand of MC1. In agreement with this observation, site-directed mutagenesis of the human MC1 recently revealed ACTH1–17 as the thermodynamically favorable ligand for this receptor (22, 54, 55). The considerable bioactivity of {alpha}-MSH2–12 in our studies confirms that a slight N- and C-terminal truncation of {alpha}-MSH does not interfere with its MC1-activating properties. Notably, we also detected ACTH2–17 as an ACTH1–39 product of EC membranes and rhuNEP.

Cleavage of ACTH1–39 at position R17-R18 and the subsequent trimming of ACTH1–17 by carboxypeptidase E activity followed by amidation and acetylation is an important step in the intracellular generation and maturation of {alpha}-MSH (3, 56). Importantly, because the digestion after R17 has been exclusively put down to activity of PC2 or related serine proteases, this is the first demonstration of an extracellular peptidase mediating this cleavage. This may partly explain the previously detected {alpha}-MSH release by HDMECs in the absence of PC2 and could be highly relevant for EC homeostasis and vascular inflammation (19, 57). Thus, rather than completely inactivating and removing ACTH and {alpha}-MSH from the extracellular space, NEP and ACE peptidolysis may generate peptides with novel MCx binding and activating properties distinct from the parental peptide. In the absence of the genuine ligands, these ACTH/{alpha}-MSH fragments could either function as MC1 agonist or may be local antagonists in the presence of the parental high-affinity peptides. They may also, as in case of KPV, even trigger cellular responses independent from known MCx.

A similar biased behavior has been proposed for the NEP substrate SP (58). Accordingly, the SP hydrolysis product SP1–7 is mimicking some but opposing other effects of the parental peptide, i.e. the central nociception (59), tumor cell migration, or cancer growth (58, 60, 61, 62). Thus, modulation of the bioactivity of neuropeptides by proteolytic products derived from the parental peptide represents a common phenomenon with not yet fully explored physiological consequences (63).

There is increasing evidence that MCx are capable of forming homo- and heterodimers (52, 64). Such GPCR dimerization and assembly with other signal transduction components frequently occurs in cholesterol-rich membrane rafts. After activation, GPCRs are internalized either in a ß-arrestin-dependent manner after traveling to clathrin-coated pits or via specialized lipid raft/calveolae microdomains of the plasma membrane (65). Importantly, cholesterol depletion impaired agonist-induced MC1 signaling in MC1-transfected ECs. Likewise, a colocalized MC1 and NEP IR was detected in low-density membrane fractions positive for membrane and lipid raft markers such as caveolin-1 and G{alpha}q. As demonstrated for ACE, NEP, and tachykinin or BK receptors, respectively, the cellular coexpression (66) or a direct sterically close association of receptor and peptidase is important for receptor function and resensitization (67, 68, 69). Thus, recruitment of protease and receptor to the same membrane microdomain may also be of functional relevance for MC1 signaling, although details of a potential association between NEP and MC1 require further clarification.

In summary, our results demonstrate that the POMC peptides ACTH and {alpha}-MSH are substrates for the Zn metalloproteases NEP and ACE, which have an important role in controlling cutaneous inflammation (29, 70) and are expressed in variable levels by dermal microvascular ECs. A local processing of ACTH1–39 or longer ACTH precursors derived from various intra- and/or extracutaneous sources, from excessive ACTH precursors present in serum (18) or even from parasites (37, 38), may give rise to smaller ACTH-related peptides, which in addition to {alpha}-MSH are potential agonist or antagonists for the MC1 and other MC receptors. Thus, proteolytic processing of POMC peptides by extracellular proteases is highly relevant for the peripheral physiological and pathophysiological regulation of pigmentary, immunomodulatory, and other responses mediated by this important class of mediators.


   Acknowledgments
 
The authors are grateful to A. Mehlich for excellent technical assistance.


   Footnotes
 
First Published Online March 15, 2007

1 T.E.S. and S.K. contributed equally to this work. Back

Abbreviations: Ab, Antibody; ACD, allergic contact dermatitis; ACE, angiotensin-converting enzyme; Ang, angiotensin; BK, bradykinin; CRE, cAMP responsive element; DNFB, 2,4-dinitro-1-fluorobenzene; EC, endothelial cell; ESI, electrospray ionization; FITC, fluorescein isothiocyanate; FSK, forskolin; GPCR, G protein-coupled receptor; HA, hemagglutinin; HDMEC, primary human dermal microvascular endothelial cell; HMEC-1, human dermal microvascular endothelial cell line 1; IBMX, 1-isobutyryl-3-methyl-xanthine; IR, immunoreactivity; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MC, melanocortin receptor; MNA, 4-methoxy-2-naphtylamin; MS, mass spectrometry; m/z, mass/charge; NEP, neprilysin (neutral endopeptidase); PA, phosphoramidon; PC, prohormone convertase; POMC; proopiomelanocortin; SP, substance P; TP, thiophan.

This work was supported by the Interdisciplinary Center of Clinical Research (to S.K.), the Deutsche Forschungsgemeinschaft (Scho 629/3-2 and Scho 629/3-P) (to T.E.S.), and Innovative Medical Research Münster (IMF SC110421) (to T.E.S. and S.K.).

Disclosure Summary: The authors have nothing to disclose.

Received December 29, 2006.

Accepted for publication March 2, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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