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The Ca²⁺-Binding Capacity of Epidermal Furin Is Disrupted by H₂O₂-Mediated Oxidation in Vitiligo

Clinical and Experimental Dermatology/Department of Biomedical Sciences (J.D.S., N.C.J.G., K.U.S.), University of Bradford, and Institute for Pigmentary Disorders in Association with the EM Arndt University Greifswald, Germany and University of Bradford, Bradford, BD7 1DP, United Kingdom; and Department of Dermatology (M.B.), University of Münster, D-41849 Münster, Germany

Address all correspondence and requests for reprints to: Professor Karin U. Schallreuter, Clinical and Experimental Dermatology, University of Bradford, Bradford BD7 1DP, United Kingdom. E-mail: k.schallreuter{at}bradford.ac.uk.

	Abstract

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The Ca²⁺-dependent precursor convertase furin is abundantly expressed in epidermal keratinocytes and melanocytes. In this context, it is noteworthy that proopiomelanocortin (POMC) cleavage is also processed by furin, leading to ACTH, β-lipotropin, and β-endorphin. All prohormone convertases including furin are regulated by Ca²⁺. Because numerous epidermal peptides and enzymes are affected by H₂O₂-mediated oxidation, including the POMC-derived peptides -MSH and β-endorphin as shown in the epidermis of patients with vitiligo, we here asked the question of whether furin could also be a possible target for this oxidation mechanism by using immunofluorescence, RT-PCR, Western blotting, Ca²⁺-binding studies, and computer modeling. Our results demonstrate significantly decreased in situ immunoreactivity of furin in the epidermis of patients with progressive vitiligo (n = 10), suggesting H₂O₂-mediated oxidation. This was confirmed by ⁴⁵Ca²⁺-binding studies with human recombinant furin identifying the loss of one Ca²⁺-binding site from the enzyme after oxidation with H₂O₂. Computer simulation supported alteration of one of the two Ca²⁺-binding sites on furin. Taken together, our results implicate that the Ca²⁺-dependent proteolytic activity of this convertase is targeted by H₂O₂, which in turn could contribute to the reduced epidermal expression of the POMC-derived peptides -MSH and β-endorphin as documented earlier in patients with vitiligo.

	Introduction

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FURIN IS A PROTEOLYTIC enzyme responsible for the conversion of precursor proteins to their biologically active peptide(s). The enzyme belongs to the Ca²⁺-dependent subtisilin-like family of precursor convertases (PC) (1). The synthesis of most PCs as inactive zymogens provides cells with the means to regulate spatially and temporally their proteolytic activities (2). This involves a number of steps, including an inhibitory mechanism preventing PC activity by blockage of the catalytic domains by their own pro-segments. Dissociation of the pro-segment and activation of the enzyme is then dependent on an increase in Ca²⁺ (3). Furin is abundantly expressed in epidermal keratinocytes at various stages of posttranslational modulation (4). The specific localization of furin, in particular the differential expression of the active site during cellular differentiation (4), together with the identification of potential prohormone convertase substrates and the Ca²⁺ dependence of all convertases strongly indicates their importance during epidermal differentiation. In addition, Berson et al. (5) recently showed that Pmel 17 (also known as gp100), which is a type I integral protein in the premelanosomal matrix of melanocytes, requires for its cleavage furin or furin-like prohormone convertases. Furin is also involved in the processing of a wide variety of substrates including albumin (6), fibrillin (7), profilaggrin (4), and Notch-1 (8), a cell-surface receptor involved in cell fate determination. It is well established that the skin has neuroendocrine capabilities that can be regulated by environmental factors (9), and the skin’s diverse immunological and inflammatory reactions are also in part under the influence of furin via mast cells (see Ref. 9). These cells release histamine in response to -MSH (10), but these cells also have the full machinery for proopiomelanocortin (POMC) cleavage including the convertase furin (11).

For a long time it was believed that the PC1/3 and PC2 convertases together with the helper protein 7B2 are primarily responsible for processing of POMC into the melanocortin peptides ACTH, - and β-MSH, the endorphins, and lipotropins (1). It was then recognized that furin and paired basic amino acid residue cleaving enzyme 4 are also capable of generating ACTH, β-lipotropin, and β-endorphin (1). Furthermore, it is well known that human epidermal cells are capable of synthesizing and processing the POMC precursor, a process that is phenotypically important for the regulation of epidermal pigmentation (12, 13). Recently, it was shown that both epidermal POMC processing as well as the derived peptides are targets for hydrogen peroxide (H₂O₂)-mediated oxidation (14) affecting the amino acid residues methionine (Met) and tryptophan (Trp) in the sequences of enzymes and peptides leading in turn to the loss of epidermal protein expression and functionality (14, 15, 16). Over the last decade, vitiligo, a depigmentation disorder of the skin, has emerged as a great model disease to follow the influence of H₂O₂-mediated oxidation because these patients accumulate up to 10^–3 M concentrations of this reactive oxygen species (ROS) in their epidermis (17). The clinical signature of vitiligo is an acquired, noncontagious idiopathic loss of the constitutive skin color from the skin that can basically occur on any part of the body at any time in life, affecting both sexes equally (18, 19). The worldwide incidence ranges from 0.5–1% (18). The cause of this ancient disease is yet unknown. Table 1 summarizes the current knowledge on the intrinsic/extrinsic sources for epidermal H₂O₂ generation in this disease and the effects on various systems identified so far. Because the presence of furin convertase has been shown in epidermal keratinocytes (4) and melanosomes of epidermal melanocytes (5), it was of interest to follow the effect of H₂O₂-mediated oxidation on furin convertase in these cells, especially because both cleavage products -MSH and β-endorphin have been shown to lose their functionality after oxidation (14). For this purpose, we used RT-PCR, Western blotting, and in situ and in vitro immunofluorescence. First, we confirmed that the protein is expressed throughout the entire epidermis, including melanocytes. We show for the first time reduced epidermal furin expression in the skin of patients with progressive vitiligo. After reduction of epidermal H₂O₂ with low-dose, narrowband UVB-activated pseudocatalase PC-KUS (Karin Ulta Schallreuter), furin levels return to normal, supporting a role for H₂O₂-dependent regulation. Specificity and stability of the antigen recognition by the antibody was proven by dot-blot analysis. We also show that furin mRNA expression is directly affected by H₂O₂ exposure (40 x 10^–6 M) already after 3 and 8 h.

In summary, the furin-dependent proteolytic action can be a target for direct oxidation by H₂O₂ under conditions of increased oxidative stress as observed in the epidermis of patients with progressive vitiligo (17). Because reduced expression and loss of functionality of the POMC peptides -MSH and β-endorphin were previously documented in this disease, our results presented herein add a novel mechanism toward the understanding of the scenario (14). Moreover, the results of this study identified yet another enzyme on the list of affected mechanisms by H₂O₂-mediated oxidation in vitiligo. It is tempting to propose that this mechanism could well occur under oxidative stress conditions in other diseases.

	Materials and Methods

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Patients and controls
For the immunofluorescence studies, we used 10 healthy controls (five male, five female) compared with patients with vitiligo. Ten patients were untreated (six male, four female) and had a progressive disease, and 10 patients underwent treatment with low-dose narrowband UVB-activated pseudocatalase PC-KUS for at least 3 months (five male, five female). All patients were of skin phototype III [Fitzpatrick classification (20)] and were age matched.

Cell culture
Epidermal melanocyte and keratinocyte cultures were established from normal human skin phototype III (Fitzpatrick classification) (20) obtained after informed consent from plastic surgery work. Whole skin samples were collected in calcium-free medium 154 (Cascade Biologics Inc., Nottinghamshire, UK) and processed within 5 h after surgery. Briefly, tissue samples were washed with 0.1 M PBS containing 12.5 µg/ml fungizone and 1% penicillin-streptomycin (Life Technologies, Inc. Invitrogen Ltd., Paisley, UK). Epidermal sheets were separated from the underlying dermis after 18 h incubation at 4 C in Dispase (Roche Applied Science, Roche Diagnostics, Indianapolis, IN). Thereafter, the epidermal sheets were trypsinized at 37 C for 20 min to form a single-cell suspension. After gentle centrifugation and resuspension of the pellet in the above media, the cells were placed in appropriate flasks and allowed to attach, changing the medium every 2–3 d. Once confluent, the cells were divided into separate melanocyte and keratinocyte cultures via selective trypsinization and the medium adapted to each cell type with the addition of calcium chloride and selected growth factors and hormones (Cascade Biologics).

Preparation of cell extracts
Epidermal cells were grown to confluence as described above before harvesting by scraping in the presence of a protease inhibitor cocktail (Amersham Biosciences, Buckinghamshire, UK) and washing the flask with 500 µl of 50 x 10^–3 M Tris buffer before centrifugation. This was followed by a repeated freeze-thaw cycle (six times) to lyse the cells. To remove any insoluble material, the sample was centrifuged again at 12,000 rpm for 10 min, and only the supernatant was subsequently used.

Determination of protein content
The protein content of cell extracts was determined spectrophotometrically at 280 nm, assuming an OD₂₈₀ of 1.0 is equal to a protein concentration of 1 mg/ml, as described by Kalb and Bernlohr (21).

In situ and in vitro immunofluorescence studies
Full skin biopsies (3 mm) were obtained after signed consent under local anesthesia from normal healthy controls (n = 10), and the lesional, nonlesional, and repigmenting skin of patients with vitiligo, with and without treatment (narrowband UVB-activated pseudocatalase PC-KUS, n = 10 from each treatment group) and stored in O.C.T. (Raymond A Lamb, East Sussex, UK) at –80 C. All probands had skin phototype III, Fitzpatrick classification (20) and were age matched. Five-micrometer-thick cryosections were cut onto poly-L-lysine-coated (Sigma, Poole, Dorset, UK) slides and were air dried at room temperature followed by fixation in ice-cold acetone for 10 min at –20 C before rehydrating in PBS for 5 min.

For in vitro immunofluorescence studies, primary confluent melanocyte and keratinocyte cultures were seeded into eight-well Lab-Tek chamber slides (ICN Biomedicals Inc., Costa Mesa, CA) and were allowed to attach. Before immunostaining, the culture medium was gently aspirated from the wells and the cells were rinsed briefly with PBS. The cells were fixed in ice-cold methanol for 10 min at –20 C and rehydrated in PBS for 5 min. Sections and cells were subsequently blocked with 10% normal donkey serum (NDS) (Serotec, Oxford, UK) diluted in PBS for 90 min at room temperature and washed with 4x PBS. Next, the slides were incubated for 2–3 h at room temperature with the primary rabbit antifurin antibody (Abcam Ltd., Cambridge, UK) diluted in 1% NDS in PBS at a dilution of 1:200 for tissue sections and 1:100 for cells. This was followed by a wash for 20 min in 4x PBS containing 0.05% Tween 20 (Sigma). To visualize the specific antigen, a fluorescein isothiocyanate-conjugated donkey antirabbit secondary antibody (Jackson Immunoresearch Laboratories Inc., West Grove, PA) was added at a 1:100 dilution in 1% NDS in PBS and incubated for 1 h at room temperature. After another wash of 4x PBS and once in 0.05% Tween 20 in PBS, the slides were once again blocked for 90 min with 10% NDS at room temperature before washing and applying the second primary antibody. The melanocyte-specific mouse anti-gp100 (NKI/beteb) monoclonal antibody (Monosan-Caltag MedSystems Ltd., Claydon, UK) was used at a dilution of 1:20 in 1% NDS in PBS or a mouse anticytokeratin 5 antibody (Cell Signaling Technology Inc., Danvers, MA) at a dilution of 1:50 for melanocytes and keratinocytes, respectively. Slides were covered and incubated at 4 C for 18 h followed by a wash in four changes of PBS and once in PBS Tween 20. A tetramethylrhodamine isothiocyanate-conjugated donkey antimouse secondary antibody (Jackson Immunoresearch) was added at a 1:100 dilution in 1% NDS in PBS and left at room temperature for 1 h. After a final wash, the slides were mounted in Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (Vector Laboratories Ltd., Peterborough, UK). For negative controls, the primary antibody was omitted and replaced with 1% NDS. Sections were viewed with a Leica DMIRB/E fluorescence microscope (Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany) and photo-documented using a digital CCD Camera, C8484-05G (Hamamatsu Photonics UK Ltd., Welwyn Garden City, UK) and the IPLab for Windows imaging software, version 3.6.4 (Scanalytics, Inc., Fairfax, VA).

RT-PCR
To determine the presence of furin mRNA in epidermal melanocytes and keratinocytes, cells were cultured as previously described and after trypsinization processed as described in the manufacturer’s protocol for the QIAGEN RNeasy Mini Kit (QIAGEN Ltd., Crawley, UK). The chosen primer sequences were 5'-TTGTAGGAGATGAGGCCACGG-3' (reverse) and 5'-GCTGGTCTTCGTCACTGTC-3' (forward) as published previously (22). PCR was performed with 1 x 10^–3 M MgCl₂ with 35 cycles of reaction, one cycle corresponding to denaturation of double-stranded DNA at 94 C for 60 sec, annealing of primers to the target sequence for 1 min at 60 C, and extension for 30 sec at 72 C. Finally, the reaction was incubated for another 10 min at 72 C to ensure full extension of all products. This step yielded the predicted product at 111 bp in length, and a negative control containing no cDNA was included.

SDS-PAGE and Western blotting
Normal human epidermal melanocyte and keratinocyte cell extracts were obtained as described above. Sample buffer (10% SDS, mercaptoethanol, glycerol, and 0.5 M Tris/HCl) was added to the supernatant before loading onto a 12% polyacrylamide gel for protein separation. After this, the polyacrylamide gel was electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) before any nonspecific binding sites were blocked by immersing the membrane in a 3% BSA (Sigma)/20 mM Tris-buffered saline with 0.047% Tween at pH 7.4 (TBS-T) blocking solution overnight at 4 C. This was followed by an incubation of 2 h with the rabbit anti-furin antibody (Abcam; 1:1000) at room temperature and a wash of 40 min in TBS-T buffer. The blot was further incubated for 1 h at room temperature with an antirabbit IgG peroxidase-conjugated antibody (Sigma; 1:5000). Visualization of the specific protein bands was performed using modified enhanced chemiluminescence fixed on a film sheet (X-OMAT; Kodak, Rochester, NY).

Dot blot
The manual spotting method used in this study followed the protocol recommended by the manufacturers of PVDF membranes (Immobilon-P; Millipore). Briefly, 1–5 µl human recombinant furin (Sigma) was applied onto a prewetted PVDF membrane, allowing the samples to be absorbed. The membrane was air dried for 15 min before blocking with 3% BSA (Sigma) in TBS-T buffer overnight at 4 C. Immunodetection of the protein followed the method described above for Western blotting. For detection of ⁴⁵Ca-bound furin, 5 µl of each fraction was applied to the membrane. Looking at the effect of oxidation on antibody recognition, 1 µl furin was applied after incubation for 2 h in the dark with different concentrations of H₂O₂ (0–100 x 10^–3 M).

⁴⁵Ca-binding assay
Human recombinant furin (Sigma) was incubated for 1 h at room temperature with 1 x 10^–3 M ⁴⁵CaCl₂ (12.2 mCi/mg; ICN Pharmaceuticals Inc., Aurora, OH) before separating the bound and free ⁴⁵Ca on a G-25 Sephadex column (GE Healthcare UK Ltd., Chalfont St. Giles, UK). Fractions (500 µl) were collected and the radioactivity of each measured in ReadySafe scintillation fluid (Beckman Coulter Inc., Fullerton, CA) on a Tri-Carb liquid scintillation analyzer (Packard-Canberra Ltd., Pangbourne, UK). The presence of protein in the fractions was examined using the dot-blot method as described above, and those fractions containing furin were pooled together. H₂O₂ (1 x 10^–3 M; Sigma) was added to the pooled fractions and left at room temperature in the dark to oxidize for 1 h before separation on a fresh G-25 Sephadex column. The fractions were collected and measured as before.

Quantitative real-time PCR
To follow the influence of H₂O₂ on mRNA expression, normal human keratinocytes were exposed for 30 min to H₂O₂ (40 x 10^–6 M) in KGM2 medium (PromoCell, Heidelberg, Germany). Cells were subsequently washed thoroughly with PBS and recultured in KGM2 medium for 3 and 8 h. Total RNA was isolated from the keratinocytes using a commercially available purification kit (QIAGEN, Hilden, Germany). After DNA digestion, samples were reverse-transcribed by oligo dT primers and RevertAid M-MuLV reverse transcriptase (Fermentas Life Sciences, Hanover, MD). Quantitative real-time PCR was performed in a total volume of 20 µl with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and with primers each at 200 x 10^–9 M. Primers were designed with the computer program Primer Express (Applied Biosystems) using parameters recommended by the manufacturer. Primer sequences for furin were 5'-GCAAAGCGACGGACTAAACG-3' (forward) and 5'-TGGAGACCACA-ATGCCGTG-3' (reverse). All reactions were done in an ABI-Prism 7300 sequence detector supplied with SDS 2.1 software (Applied Biosystems) in duplicates, using the following conditions: an initial activation step of 2 min at 50 C and a single denaturation step of 15 min at 95 C followed by 40 cycles of 15 sec at 95 C and 60 sec at 60 C and a final cycle of 15 sec at 95 C, 15 sec at 60 C, and 15 sec 95 C. Gene expression levels were determined by using the 2^–CT method (23) employing GAPDH gene expression as an endogenous control. The GAPDH primers were 5'-TGCACCACCA-ACTGCTTAGC-3' (forward) and 5'-GGCATGGACTGTGGTCATGAG-3' (reverse).

Molecular modeling
The crystal structure of furin was obtained from the Protein Data Bank (1P8J) (24). All Met and Trp residues were oxidized to methionine sulfoxide and hydroxytryptophan and then minimized using HyperChem software (Hypercube Inc., Gainesville, FL). The resulting structure was analyzed by Deep View (Swiss Institute of Bioinformatics, Basel, Switzerland), and structural changes were compared with the native structure of the enzyme.

Statistics
Student’s t test was used to test the significance of the differences in mRNA expression. For each condition, the ground condition was set as 1. Expression levels were depicted as mean expression values ± SD. For image analysis of staining intensities, we used the two-tailed unpaired t test to evaluate the differences between control and patient sections.

The study was approved by the local Ethics Committee and was in agreement with the Helsinki declaration.

	Results

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In situ and in vitro protein expression confirmed the presence of furin throughout the epidermis, including melanocytes
Previous studies documented the presence of furin in epidermal keratinocytes and in melanosomes of melanocytes (4, 5). Our in situ results confirm those findings. In addition, we show the presence of furin in epidermal melanocytes in situ (Fig. 1A). The expression of furin is also documented in epidermal keratinocytes and melanocytes under in vitro conditions (Fig. 1, B and C).

View larger version (23K): [in this window] [in a new window]   FIG. 1. In situ and in vitro furin expression in the human epidermis. A, Healthy control (skin phototype III, Fitzpatrick classification) showing the presence of furin throughout the epidermis. Overlay with the melanocyte-specific gp100 protein (NKI/beteb, red/yellow) identifies the presence of furin in melanocytes (arrows). B, Epidermal melanocytes express furin throughout the cell under in vitro conditions. C, Epidermal keratinocytes express furin throughout the cell under in vitro conditions. Magnification, x400. Identity of cells were confirmed with NKI/beteb and cytokeratin 5 markers for melanocytes and keratinocytes, respectively (data not shown).

View larger version (43K): [in this window] [in a new window]   FIG. 2. Furin is present in epidermal cells at the mRNA and protein level. A, RT-PCR identifies furin mRNA in epidermal melanocytes (MC) and keratinocytes (KC). The positive bands at 111 bp are in agreement with the presence of furin mRNA obtained via RT-PCR using primer pairs previously established by Böhm et al. (22 ). B, SDS-PAGE and Western blotting identifies immunopositive bands for furin in melanocyte (MC) and keratinocyte (KC) cell extracts. The band at 59 kDa is predicted to correspond to the processed proteins after proregion cleavage.

Low epidermal furin expression in vitiligo returns to normal after reduction of H₂O₂ concentrations by a pseudocatalase PC-KUS
Because patients with vitiligo accumulate 10^–3 M H₂O₂ as shown in vivo by Fourier Transform Raman spectroscopy, we wanted to know whether furin expression was affected by this ROS (17). Our results show that furin expression is significantly reduced (P < 0.0005 and P < 0.005) in both the lesional and nonlesional skin of untreated patients with progressive vitiligo compared with healthy controls (Fig. 3). After reduction of these high H₂O₂ levels by a narrowband UVB-activated pseudocatalase PC-KUS (27), furin levels return to normal in nonlesional skin, whereas in actively repigmenting skin, the levels are significantly increased (P < 0.005) (Fig. 3).

View larger version (45K): [in this window] [in a new window]   FIG. 3. Epidermal expression of furin is significantly decreased in the skin of patients with progressive vitiligo (+ H2O2) compared with healthy controls and returns to normal levels after treatment by reduction of H2O2 with a narrowband UVB-activated pseudocatalase PC-KUS (– H2O2). Images of the immunoreactivity were taken at a magnification of x400 in healthy control skin (n = 10) and compared with uninvolved skin (i) (NL) (n = 10), lesional skin (ii) (VIT) (n = 10), and repigmenting skin (iii) (n = 10) of patients with vitiligo. For image analysis of the staining intensities (inset), the IPLab imaging software was used. ***, P < 0.0005; **, P < 0.005; ns, nonsignificant, P > 0.025.

H₂O₂ oxidation affects furin at the mRNA level
Furin mRNA from normal human keratinocytes was measured before and after incubation of the cells over 30 min with 40 x 10^–6 M H₂O₂. The effect was tested after 3 and 8 h. Expression of furin mRNA is significantly increased in the presence of H₂O₂ after 3 h (1.8-fold) and 8 h (1.3-fold) (data not shown). This observation seems to suggest that mRNA is induced by 10^–6 M levels of H₂O₂, possibly explaining the observed increase in epidermal furin expression in the repigmenting skin of patients with vitiligo after reduction of 10^–3 M H₂O₂ levels by pseudocatalase PC-KUS. Whether higher concentrations affect mRNA directly needs to be shown because under physiological conditions, the cell degrades any excess H₂O₂ via catalase and other enzymes to keep homeostasis in place.

⁴⁵Ca²⁺-binding studies reveal the loss of one Ca²⁺-binding site from furin after H₂O₂-mediated oxidation
Although dot-blot analysis showed that the antibody binding epitope of furin was unaffected by H₂O₂, we cannot dismiss that furin could be oxidized. To follow the effect of H₂O₂ on the Ca²⁺-binding potential of human recombinant furin, we used ⁴⁵Ca²⁺ to examine binding of this ion before and after oxidation with H₂O₂ (1 x 10^–3 M). The results showed a decrease of 55% after oxidation (Fig. 4). Furin-bound ⁴⁵Ca²⁺ eluted in fractions 5–8, which was confirmed by detection of the furin protein via dot-blot analysis (inset). The corresponding unbound ⁴⁵Ca²⁺ eluted at fraction 12. This result implies a loss of half of the proteolytic activity under oxidizing conditions. The kinetics for ⁴⁵Ca binding to the EF-hand sites on furin could not be determined due to the fast exchange of this ion (10^–3 sec) on the binding sites (28, 29).

View larger version (24K): [in this window] [in a new window]   FIG. 4. Oxidation by H2O2 reduces the binding of 45Ca2+ to human recombinant furin by 55%. Furin-bound 45Ca2+ before () and after () oxidation was placed on a G-25 Sephadex column. The eluted fractions were measured for radioactivity. The protein-bound 45Ca2+ eluted in fractions 5–8 (confirmed by dot-blot analysis, inset), whereas the unbound 45Ca eluted after fraction 12 (arrow).

View larger version (55K): [in this window] [in a new window]   FIG. 5. HyperChem molecular modeling of the crystal structure of native and oxidized furin identifies the loss of one Ca2+ from binding site 2 in the enzyme active site. The calcium binding site 2 of furin is located in the enzyme active site, whereas calcium binding site 1 is located in the N terminus of the enzyme. Applying computer simulation in the presence and absence of H2O2 allows identification of structural alterations. A, The substrate binding (active site) of furin contains the catalytic triad of Asp153, His194, and Ser368 (native enzyme). After oxidation of Met and Trp residues in the sequence with H2O2, the H-bond between Asp153 and His194 remains intact, but the other H-bond between His194 and Ser368 is lost, resulting in disruption of the catalytic triad (oxidized enzyme). This result would suggest loss of enzyme activity. B, In the native Ca2+-binding site 2 of the enzyme active site, this ion is coordinated by O-donor atoms from only three amino acids (Asp258, Asp301, and Glu331) and by three internal H2O molecules. Oxidation by H2O2 results in significantly increased distances for all three amino acids and H2O molecules yielding a complete loss of the Ca2+ ion from its binding domain. C, In the native Ca2+-binding site 1, Ca2+ is coordinated by O-donors from six amino acids (Asp115, Asn208, Asp162, Val205, Val210, and Gly212). Oxidation by H2O2 results in increased distances for Asp115, Asp162, and Gly212. However, this would lead only to a small change in this Ca2+-binding site, which is not affecting the Ca2+ binding. Taken together, the result from computer modeling supports the results from the 45Ca-binding study as shown in Fig. 4. Color code: red, acidic amino acid O-donors; yellow, H2O molecules; green ball, calcium; blue, -helices; green arrows, pleated sheets; beige, linking sequences.

	Discussion

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Human epidermal keratinocytes and melanocytes have the full capacity for POMC processing by the prohormone convertases PC1 and PC2/7B2 (see Ref. 9). In epidermal melanocytes, POMC processing via PC1 and PC2/7B2 as well as furin has been shown to occur primarily in the melanosome (5, 30, 31). In fact, melanocytes express significantly higher levels of POMC than keratinocytes both in situ and in vitro (30, 31). In this report, we confirm in situ and in vitro that epidermal keratinocytes and melanocytes express the convertase furin (Fig. 1). The positive immunoreactivity is supported by furin mRNA expression (RT-PCR) and by Western blot analyses (Fig. 2). Because the presence of furin has been shown in melanosomes (5), this enzyme could contribute to the direct regulation of melanocyte function and epidermal pigmentation via POMC-derived melanocortins.

As said above, patients with the depigmentation disorder vitiligo suffer from severe oxidative stress due to overproduction of H₂O₂ leading to accumulation of 10^–3 M H₂O₂ in their epidermis (Table 1). Therefore, this disease has emerged as an excellent model to study the influence of H₂O₂-mediated stress in vitro and in vivo (32). In this context, it has been shown that this constant ongoing oxidative stress leads to decreased activities of many enzymes, and it affects several peptides including the melanocortins ACTH, -MSH, and β-endorphin (Table 1) (14, 33). Here we demonstrate that furin expression is also a potential target for oxidation by H₂O₂ in the epidermis of these patients. In fact, oxidation of one Ca²⁺-binding site decreases the binding potential by 55%. Ca²⁺ binding is also severely affected in the important calcium-binding protein calmodulin where H₂O₂-mediated oxidation affects all four EF-hand binding sites of the protein (16). The loss of Ca²⁺ from calmodulin seriously affects calmodulin-dependent calcium ATPase activity in the skin of patients with vitiligo (16) which in turn jeopardizes L-phenylalanine uptake and its turnover to L-tyrosine in epidermal cells (16). Hence, the result presented herein adds another mechanism to the documented impaired epidermal Ca²⁺ homeostasis in vitiligo that was already recognized earlier in both melanocytes and keratinocytes established from lesional skin of these patients (34).

In the light of POMC cleavage/homeostasis, it is noteworthy that all prohormone convertases (PC1, PC2, furin, and paired basic amino acid residue cleaving enzyme 4) have two Ca²⁺-binding domains, where Ca²⁺-binding site 1 is important for the stability of these proteases, but Ca²⁺-binding site 2 is crucial for the active site structure (26). Our results with furin support that only Ca²⁺-binding site 2 loses its Ca²⁺ ion, implying reduced furin activity in progressive vitiligo. Whether this is also the case for the other convertases needs to be shown. The computer model predicts alteration by H₂O₂-mediated oxidation in all of them. Unfortunately, these proteins are not yet available to pursue binding studies with radiolabeled Ca²⁺.

Taken together, our data provide further support for the observed decrease in POMC processing in vitiligo (14, 33). Importantly, these results suggest a novel general mechanism for all prohormone convertases, implying that POMC cleavage is sensitive to H₂O₂-mediated stress via their Ca²⁺-binding domains in the active site of these enzymes.

	Footnotes

 
This research was supported by Stiefel International with a grant to K.U.S.

The results were part of a Ph.D. thesis, University of Bradford, UK (J.D.S.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 3, 2008

Abbreviations: KUS, Karin Ulta Schallreuter; NDS, normal donkey serum; PC, precursor convertases; POMC, proopiomelanocortin; PVDF, polyvinylidene difluoride; ROS, reactive oxygen species; TBS-T, Tris-buffered saline with 0.047% Tween at pH 7.4.

Received September 24, 2007.

Accepted for publication December 26, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Seidah NG, Benjannet S, Hamelin J, Mamarbachi AM, Basak A, Marcinkiewicz J, Mbikay M, Chretien M, Marcinkiewicz M 1999 The subtilisin/kexin family of precursor convertases. Emphasis on PC1, PC2/7B2, POMC and the novel enzyme SKI-1. Ann NY Acad Sci 885:57–74[Medline]
2. Khan AR, James MN 1998 Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes. Protein Sci 7:815–836[Medline]
3. Anderson ED, VanSlyke JK, Thulin CD, Jean F, Thomas G 1997 Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage. EMBO J 16:1508–1518[CrossRef][Medline]
4. Pearton DJ, Nirunsuksiri W, Rehemtulla A, Lewis SP, Presland RB, Dale BA 2001 Proprotein convertase expression and localization in epidermis: evidence for multiple roles and substrates. Exp Dermatol 10:193–203[CrossRef][Medline]
5. Berson JF, Theos AC, Harper DC, Tenza D, Raposo G, Marks MS 2003 Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis. J Cell Biol 161:521–533[Abstract/Free Full Text]
6. Mori K, Imamaki A, Nagata K, Yonetomi Y, Kiyokage-Yoshimoto R, Martin TJ, Gillespie MT, Nagahama M, Tsuji A, Matsuda Y 1999 Subtilisin-like proprotein convertases, PACE4 and PC8, as well as furin, are endogenous proalbumin convertases in HepG2 cells. J Biochem (Tokyo) 125:627–633[Abstract/Free Full Text]
7. Raghunath M, Putnam EA, Ritty T, Hamstra D, Park ES, Tschodrich-Rotter M, Peters R, Rehemtulla A, Milewicz DM 1999 Carboxy-terminal conversion of profibrillin to fibrillin at a basic site by PACE/furin-like activity required for incorporation in the matrix. J Cell Sci 112(Pt 7):1093–1100
8. Logeat F, Bessia C, Brou C, LeBail O, Jarriault S, Seidah NG, Israel A 1998 The Notch1 receptor is cleaved constitutively by a furin-like convertase. Proc Natl Acad Sci USA 95:8108–8112[Abstract/Free Full Text]
9. Slominski A, Wortsman J 2000 Neuroendocrinology of the skin. Endocr Rev 21:457–487[Abstract/Free Full Text]
10. Grutzkau A, Henz BM, Kirchhof L, Luger T, Artuc M 2000 -Melanocyte stimulating hormone acts as a selective inducer of secretory functions in human mast cells. Biochem Biophys Res Commun 278:14–19[CrossRef][Medline]
11. Artuc M, Böhm M, Grutzkau A, Smorodchenko A, Zuberbier T, Luger T, Henz BM 2006 Human mast cells in the neurohormonal network: expression of POMC, detection of precursor proteases, and evidence for IgE-dependent secretion of -MSH. J Invest Dermatol 126:1976–1981[CrossRef][Medline]
12. Slominski A, Wortsman J, Luger T, Paus R, Solomon S 2000 Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress. Physiol Rev 80:979–1020[Abstract/Free Full Text]
13. Slominski A, Tobin DJ, Shibahara S, Wortsman J 2004 Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 84:1155–1228[Abstract/Free Full Text]
14. Spencer JD, Gibbons NC, Rokos H, Peters EM, Wood JM, Schallreuter KU 2007 Oxidative stress via hydrogen peroxide affects proopiomelanocortin peptides directly in the epidermis of patients with vitiligo. J Invest Dermatol 127:411–420[CrossRef][Medline]
15. Gibbons NC, Wood JM, Rokos H, Schallreuter KU 2006 Computer simulation of native epidermal enzyme structures in the presence and absence of hydrogen peroxide (H₂O₂): potential and pitfalls. J Invest Dermatol 126:2576–2582[CrossRef][Medline]
16. Schallreuter KU, Gibbons NC, Zothner C, Abou Elloof MM, Wood JM 2007 Hydrogen peroxide-mediated oxidative stress disrupts calcium binding on calmodulin: more evidence for oxidative stress in vitiligo. Biochem Biophys Res Commun 360:70–75[CrossRef][Medline]
17. Schallreuter KU, Moore J, Wood JM, Beazley WD, Gaze DC, Tobin DJ, Marshall HS, Panske A, Panzig E, Hibberts NA 1999 In vivo and in vitro evidence for hydrogen peroxide H₂O₂ accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase. J Investig Dermatol Symp Proc 4:91–96[Medline]
18. Ortonne JP, Bose SK 1993 Vitiligo: where do we stand? Pigment Cell Res 6:61–72[CrossRef][Medline]
19. Schallreuter KU 2005 Vitiligo. In: Hertl M, ed. Autoimmune diseases of the skin: pathogenesis, diagnosis, management. 2nd ed. Vienna: Springer; 367–384
20. Fitzpatrick TB, Miyamoto M, Ishikawa K 1967 The evolution of concepts of melanin biology. Arch Dermatol 96:305–323[Abstract/Free Full Text]
21. Kalb Jr VF, Bernlohr RW 1977 A new spectrophotometric assay for protein in cell extracts. Anal Biochem 82:362–371[CrossRef][Medline]
22. Böhm M, Eickelmann M, Li Z, Schneider SW, Oji V, Diederichs S, Barsh GS, Vogt A, Stieler K, Blume-Peytavi U, Luger TA 2005 Detection of functionally active melanocortin receptors and evidence for an immunoregulatory activity of -melanocyte-stimulating hormone in human dermal papilla cells. Endocrinology 146:4635–4646[Abstract/Free Full Text]
23. Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(–C_T) method. Methods 25:402–408[CrossRef][Medline]
24. Henrich S, Cameron A, Bourenkov GP, Kiefersauer R, Huber R, Lindberg I, Bode W, Than ME 2003 The crystal structure of the proprotein processing proteinase furin explains its stringent specificity. Nat Struct Biol 10:520–526[CrossRef][Medline]
25. Bassi DE, Fu J, Lopez de Cicco R, Klein-Szanto AJ 2005 Proprotein convertases: "master switches" in the regulation of tumor growth and progression. Mol Carcinog 44:151–161[CrossRef][Medline]
26. Henrich S, Lindberg I, Bode W, Than ME 2005 Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity. J Mol Biol 345:211–227[CrossRef][Medline]
27. Schallreuter KU 1999 Successful treatment of oxidative stress in vitiligo. Skin Pharmacol Appl Skin Physiol 12:132–138[CrossRef][Medline]
28. Tufty RM, Kretsinger RH 1975 Troponin and parvalbumin calcium binding regions predicted in myosin light chain and T4 lysozyme. Science 187:167–169[Abstract/Free Full Text]
29. Kretsinger RH, Tolbert D, Nakayama S, Pearson W 1991 The EF-hand, homologs and analogs. In: Heizmann C, ed. Novel calcium-binding proteins: fundamentals and clinical implications. Berlin: Springer-Verlag; 17–37
30. Peters EM, Tobin DJ, Seidah NG, Schallreuter KU 2000 Pro-opiomelanocortin-related peptides, prohormone convertases 1 and 2 and the regulatory peptide 7B2 are present in melanosomes of human melanocytes. J Invest Dermatol 114:430–437[CrossRef][Medline]
31. Kauser S, Schallreuter KU, Thody AJ, Gummer C, Tobin DJ 2003 Regulation of human epidermal melanocyte biology by β-endorphin. J Invest Dermatol 120:1073–1080[CrossRef][Medline]
32. Schallreuter KU 2005 Vitiligo. In: Hertl M, ed. Autoimmune diseases of the skin: pathogenesis, diagnosis, management. 2nd ed. Vienna: Springer; 367–384
33. Graham A, Westerhof W, Thody AJ 1999 The expression of -MSH by melanocytes is reduced in vitiligo. Ann NY Acad Sci 885:470–473[Medline]
34. Schallreuter-Wood KU, Pittelkow MR, Swanson NN 1996 Defective calcium transport in vitiliginous melanocytes. Arch Dermatol Res 288:11–13[CrossRef][Medline]
35. Schallreuter KU, Wood JM, Pittelkow MR, Büttner G, Swanson N, Körner C, Ehrke C 1996 Increased monoamine oxidase A activity in the epidermis of patients with vitiligo. Arch Dermatol Res 288:14–18[Medline]
36. Schallreuter KU 1999 A review of recent advances on the regulation of pigmentation in the human epidermis. Cell Mol Biol (Noisy-le-grand) 45:943–949[Medline]
37. Darr D, Fridovich I 1994 Free radicals in cutaneous biology. J Invest Dermatol 102:671–675[CrossRef][Medline]
38. Moretti S, Spallanzani A, Amato L, Hautmann G, Gallerani I, Fabiani M, Fabbri P 2002 New insights into the pathogenesis of vitiligo: imbalance of epidermal cytokines at sites of lesions. Pigment Cell Res 15:87–92[CrossRef][Medline]
39. Wong GH, Goeddel DV 1988 Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science 242:941–944[Abstract/Free Full Text]
40. Schallreuter KU, Rokos H, Chavan B, Gillbro JM, Cemeli E, Zothner C, Anderson D, Wood JM 2008 Quinones are reduced by 6-tetrahydrobiopterin in human keratinocytes, melanocytes, and melanoma cells. Free Radic Biol Med 44:538–546[CrossRef][Medline]
41. Rokos H, Beazley WD, Schallreuter KU 2002 Oxidative stress in vitiligo: photo-oxidation of pterins produces H₂O₂ and pterin-6-carboxylic acid. Biochem Biophys Res Commun 292:805–811[CrossRef][Medline]
42. Anderson D, Schmid TE, Baumgartner A, Cemeli-Carratala E, Brinkworth MH, Wood JM 2003 Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay). Mutat Res 544:173–178[CrossRef][Medline]
43. Thöny B, Auerbach G, Blau N 2000 Tetrahydrobiopterin biosynthesis, regeneration and functions. Biochem J 347(Pt 1):1–16
44. Schallreuter KU, Wood JM, Pittelkow MR, Gütlich M, Lemke KR, Rödl W, Swanson NN, Hitzemann K, Ziegler I 1994 Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin. Science 263:1444–1446[Abstract/Free Full Text]
45. Schallreuter KU, Moore J, Wood JM, Beazley WD, Peters EM, Marles LK, Behrens-Williams SC, Dummer R, Blau N, Thöny B 2001 Epidermal H₂O₂ accumulation alters tetrahydrobiopterin (6BH₄) recycling in vitiligo: identification of a general mechanism in regulation of all 6BH₄-dependent processes? J Invest Dermatol 116:167–174[CrossRef][Medline]
46. Shalbaf M, Gibbons NC, Wood JM, Rokos H, Elwary SM, Marles LK, Schallreuter KU, The presence of epidermal allantoin further supports oxidative stress in vitiligo. Exp Dermatol, in press
47. Arnoff S 1965 Catalase: kinetics of photo-oxidation. Science 150:72–73[Abstract/Free Full Text]
48. Maresca V, Flori E, Briganti S, Camera E, Cario-Andre M, Taieb A, Picardo M 2006 UVA-induced modification of catalase charge properties in the epidermis is correlated with the skin phototype. J Invest Dermatol 126:182–190[CrossRef][Medline]
49. Wood JM, Schallreuter KU 2006 UVA-irradiated pheomelanin alters the structure of catalase and decreases its activity in human skin. J Invest Dermatol 126:13–14[CrossRef][Medline]
50. Schallreuter KU, Wood JM 2001 Thioredoxin reductase: its role in epidermal redox status. J Photochem Photobiol B 64:179–184[CrossRef][Medline]
51. Beazley WD, Gaze D, Panske A, Panzig E, Schallreuter KU 1999 Serum selenium levels and blood glutathione peroxidase activities in vitiligo. Br J Dermatol 141:301–303[CrossRef][Medline]
52. Schallreuter K, Gibbons NCJ, Rokos H, Ruebsam K, Wood JM, Hydrogen peroxide mediated oxidation severely compromises the methionine sulfoxide A/thioredoxin reductase antioxidant defence system. Proc 36th Annual European Society for Dermatological Research Meeting, Paris, 2006, p s15
53. Rokos HM J, Hasse S, Gillbro JM, Wood JM, Schallreuter KU 2004 In vivo fluorescence excitation spectroscopy and in vivo Fourier-transform Raman spectroscopy in human skin: evidence of H₂O₂ oxidation of epidermal albumin in patients with vitiligo. J Raman Spectrosc 35:125–130[CrossRef]
54. Schallreuter KU, Chavan B, Rokos H, Hibberts N, Panske A, Wood JM 2005 Decreased phenylalanine uptake and turnover in patients with vitiligo. Mol Genet Metab 86(Suppl 1):S27–S33
55. Wood JM, Chavan B, Hafeez I, Schallreuter KU 2005 Regulation of tyrosinase by tetrahydropteridines–what is real? A critical reanalysis of H. Wojtasek’s view. Biochem Biophys Res Commun 331:891–893[CrossRef][Medline]
56. Schallreuter KU, Elwary SM, Gibbons NC, Rokos H, Wood JM 2004 Activation/deactivation of acetylcholinesterase by H₂O₂: more evidence for oxidative stress in vitiligo. Biochem Biophys Res Commun 315:502–508[CrossRef][Medline]
57. Schallreuter KU, Gibbons NC, Zothner C, Elwary SM, Rokos H, Wood JM 2006 Butyrylcholinesterase is present in the human epidermis and is regulated by H2O2: more evidence for oxidative stress in vitiligo. Biochem Biophys Res Commun 349:931–938[CrossRef][Medline]
58. Hasse S, Gibbons NC, Rokos H, Marles LK, Schallreuter KU 2004 Perturbed 6-tetrahydrobiopterin recycling via decreased dihydropteridine reductase in vitiligo: more evidence for H₂O₂ stress. J Invest Dermatol 122:307–313[CrossRef][Medline]
59. Schallreuter KU, Elwary S 2007 Hydrogen peroxide regulates the cholinergic signal in a concentration dependent manner. Life Sci 80:2221–2226[CrossRef][Medline]

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