Production of the Soluble Form of KIT, s-KIT, Abolishes Stem Cell Factor-Induced Melanogenesis in Human Melanocytes

ORIGINAL ARTICLE

Production of the Soluble Form of KIT, s-KIT, Abolishes Stem Cell Factor-Induced Melanogenesis in Human Melanocytes Shinya Kasamatsu1, Akira Hachiya1,2, Kazuhiko Higuchi1, Atsushi Ohuchi1, Takashi Kitahara1 and Raymond E. Boissy3,4 The signaling of stem cell factor (SCF) and its receptor KIT (membrane-bound KIT; m-KIT) plays an important role in melanocyte development, survival, proliferation, and melanogenesis. It has been demonstrated in other systems that a soluble form of m-KIT released from the cell surface (s-KIT) regulates SCF signaling, although there have been no reports pertaining to the existence and the biological role of s-KIT in melanocytes. In this study, we therefore examined the involvement of s-KIT in melanogenesis. Western blotting analysis revealed that treatment with phorbol 12-myristate-13-acetate (PMA) or 4-aminophenylmercuric acetate (APMA) induced s-KIT production in cultured human melanocytes. Inhibitors of tumor necrosis factor-a-converting enzyme (TACE) and metalloproteinases (MMPs) muted this release of s-KIT into the media. Human recombinant s-KIT added to melanocytes inhibited SCF-induced phosphorylation of m-KIT, resulting in suppression of SCFinduced melanogenesis. Additionally, APMA-induced s-KIT production abolished SCF-induced melanogenesis as effectively as a KIT-neutralizing antibody. Concomitantly, APMA and TACE inhibitors significantly decreased and increased melanin synthesis, respectively, in an in vitro skin model. Taken together, these findings provided an insight into the elaborate mechanism of SCF/m-KIT signaling in human melanocytes and suggested that production of s-KIT contributes to the regulation of human skin pigmentation. Journal of Investigative Dermatology (2008) 128, 1763–1772; doi:10.1038/jid.2008.9; published online 31 January 2008

INTRODUCTION The receptor tyrosine kinase membrane-bound KIT protein (m-KIT) and its ligand stem cell factor (SCF), also referred to as steel factor, kit ligand or mast cell growth factor, are encoded by the dominant white spotting (W) locus and the steel (Sl) locus, respectively. Mutations in either of those loci elicit similar phenotypes characterized by the loss of neural crest-derived pigment cells, hematopoietic cells, and primordial germ cells (Matsui et al., 1990; Orr-Urtreger et al., 1990; Bernstein et al., 1991; Williams et al., 1992; Besmer et al., 1993; Halaban and Moellmann, 1993; Galli et al., 1994), suggesting that SCF/m-KIT signaling is essential for survival of 1

Kao Biological Science Laboratories, Tochigi, Japan; 2Department of Dermatology, Tokyo Medical University, Tokyo, Japan; 3The Skin Sciences Institute, Cincinnati Children’s Hospital Research Foundation, Cincinnati, Ohio, USA and 4Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA Correspondence: Dr Raymond E. Boissy, Department of Dermatology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, ML 592, Cincinnati, Ohio 45267-0592, USA. E-mail: [email protected] Abbreviations: APMA, 4-aminophenylmercuric acetate; 3D, three dimensional; FGF, fibroblast growth factor; m-KIT, membrane-bound KIT protein; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate-13-acetate; SCF, stem cell factor; s-KIT, soluble form of KIT; TACE, tumor necrosis factor-a-converting enzyme; TAPI-1, tumor necrosis factor-a protease inhibitor

Received 11 June 2007; revised 7 November 2007; accepted 14 November 2007; published online 31 January 2008

& 2008 The Society for Investigative Dermatology

these specific cell types. The involvement of SCF/m-KIT signaling in melanocyte development during embryogenesis has been demonstrated using phenotype analysis of Sl and W mice. In addition, experiments using a monoclonal KIT antibody (ACK2), an antagonistic blocker of m-KIT function, also revealed an important role of SCF/m-KIT signaling in the development of murine melanocytes (Nishikawa et al., 1991; Okura et al., 1995; Yoshida et al., 1996). Mutations in the human genes encoding KIT results in piebaldism, a congenic disorder that is characterized by amelanotic patches on acral and/or ventral skin surfaces, but apparently lacking detectable defects in germ cells or in the hematologic system (Giebel and Spritz, 1991; Spritz et al., 1992; Ezoe et al., 1995). In addition, intradermal injection of SCF enhances the number, size, and dendricity of melanocytes in normal human skin xenografts, whereas interruption of SCF binding to its receptor m-KIT by the injection of KITneutralizing antibody decreases these parameters (Grichnik et al., 1998). Furthermore, SCF/m-KIT signaling also mediates UVB-induced pigmentation and several pigmentation disorders. We previously reported that UVB irradiation augments gene and protein expression of membrane-bound SCF in epidermal keratinocyte to activate neighboring melanocyte via m-KIT receptors, and injection of KIT-inhibitory antibody abolishes UVB-induced pigmentation on dorsal skin of pigmented guinea pig (Hachiya et al., 2001). In the human epidermis of lentigo seniles, SCF expression is increased www.jidonline.org 1763

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compared with adjacent normal skin (Hattori et al., 2004). In dermatofibroma with epidermal hyperpigmentation, SCF secreted by dermal fibroblasts stimulates melanocytes located in the epidermis overlying the dermal fibroblast tumor (Shishido et al., 2001). These lines of evidence clearly demonstrate the role of SCF/m-KIT signaling in regulating epidermal melanogenesis under homeostatic, stimulatory, or pathogenic conditions (including UVB exposure and pigmentation disorders). SCF is expressed as both an 18-kDa soluble and a 31-kDa membrane-bound form, whereas m-KIT is expressed as a 145-kDa glycosylated transmembrane protein and consists of an extracellular, a transmembrane, and a tyrosine kinase domain (Broudy, 1997; Zhang et al., 2000; Ronnstrand, 2004; Lennartsson et al., 2005). Moreover, the extracellular domain of m-KIT was found to consist of five Ig-like domains, with the first three Ig-like domains and the fourth domain involved in binding SCF and receptor dimerization, respectively (Blechman et al., 1993, 1995; Lev et al., 1993; Zhang et al., 2000). Binding and dimerization subsequently cause autophosphorylation at tyrosine residues of m-KIT, followed by the activation of downstream signaling cascades (Blume-Jensen et al., 1991; Ronnstrand, 2004; Lennartsson et al., 2005). In mast cells, hematopoietic cells, and endothelial cells, release of the fifth Ig-like extracellular domain of m-KIT (called soluble form of KIT (s-KIT)), corresponding to 98–100-kDa glycoprotein, is induced by stimulants such as phorbol 12-myristate-13-acetate (PMA) (Yee et al., 1993; Broudy et al., 1994, 2001; Turner et al., 1995). One of the mechanisms underlying the cleavage of s-KIT from m-KIT by PMA is considered to be via activation of protein kinase C (Yee et al., 1993, 1994). Recently, tumor necrosis factor-a-converting enzyme (TACE) and some matrix metalloproteinases (MMPs) were also found to catalyze the production of s-KIT in mast cells (Cruz et al., 2004). Metalloproteinases activated by divalent metal ions are classified into two groups, one consisting of membranebound type metalloproteinases, called a disintegrin and metalloproteinase domain family, and the other consisting of MMPs (Arribas and Borroto, 2002). TACE belongs to the disintegrin and metalloproteinase domain family and has approximately 20 substrates such as tumor necrosis factor-a, transforming growth factor-b, Notch, as well as m-KIT (Mezyk et al., 2003; Cruz et al., 2004). TACE is activated by PMA via activation of protein kinase C, resulting in the production of s-KIT (Doedens et al., 2003; Cruz et al., 2004). In addition, the mercurial compound 4-aminophenylmercuric acetate (APMA) activates TACE and MMPs (Merlos-Suarez et al., 2001); however, the precise mechanisms of action have not been delineated. s-KIT has been detected in vivo in human serum or plasma (Wypych et al., 1995). The amount of s-KIT is increased in serum of patients with mastocytosis and correlates with disease severity, implicating s-KIT as a potential molecular marker of the disease (Akin et al., 2000; Kanbe et al., 2001). Additionally, the amount of s-KIT in serum correlates with leukemic cell burden, graft-versus-host disease, and delayed engraftment following bone marrow transplantation (Hashino 1764 Journal of Investigative Dermatology (2008), Volume 128

et al., 1995, 1997; Kawakita et al., 1995; Tajima et al., 1998). Of note, s-KIT has an ability to bind SCF and functions as a decoy receptor to inhibit SCF/m-KIT signaling (Dahlen et al., 2001). Therefore, these data indicate a potential role of s-KIT in regulation of SCF/m-KIT signaling in m-KIT-harboring cells, including melanocyte, even though released s-KIT from melanocytes has not been reported. In this study, we demonstrate that s-KIT is expressed by human melanocytes in vitro and in the human epidermis in vivo. The production of s-KIT is enhanced by stimulants such as PMA, TACE, and MMP activators. We further demonstrate that s-KIT itself or release of s-KIT could inhibit SCF-induced melanogenesis in human melanocytes. RESULTS Detection of KIT in soluble and membrane types in human epidermis in vivo and in human melanocytes in vitro

On the basis of the information that the production of a soluble form of KIT protein is identified as the appearance 100-kDa protein, whereas an m-KIT protein is identified as a 145-kDa protein, in mast and hematopoietic cells (Yee et al., 1993; Turner et al., 1995), we investigated whether or not the ectodomain of m-KIT of human melanocytes was cleaved endogenously. Western blotting analysis using an KIT N-terminus-specific antibody revealed that a significant amount of s-KIT and m-KIT, at approximately 100 and 145-kDa, respectively, was detected in the protein extracts from human epidermal sheets, suggesting a continuous production of s-KIT in human epidermis in vivo (Figure 1a) Subsequently, given that the cited literature also reported that PMA induces a cleavage of s-KIT (Yee et al., 1993; Broudy et al., 2001), this stimulant was used in this study to investigate the induction of s-KIT from human melanocytes. Western blotting analyses demonstrated differences in molecular weight between s-KIT and m-KIT in cultured supernatant of human melanocytes treated by PMA for 4 hours and non-treated melanocyte lysate, respectively (Figure 1b). In addition, s-KIT production by PMA was found to be increased in a time-dependent manner, accompanied by concomitant decrease in the expression of m-KIT, suggesting that s-KIT is cleaved from m-KIT in human melanocytes in vitro (Figure 2). s-KIT inhibited SCF-induced phosphorylation in human melanocytes

After observing the production of s-KIT in human melanocytes, we investigated whether human recombinant s-KIT inhibits the binding of SCF to m-KIT receptor on melanocytes. SCF-binding assay demonstrated that human recombinant s-KIT inhibited the binding of 125I-labeled SCF to m-KIT on human melanocytes in a dose-dependent manner (Figure 3a), suggesting a role of s-KIT as a decoy receptor in human melanocytes, consistent with a previous report on KITtransfected mast cells (Dahlen et al., 2001). To further explore the inhibitory effect of s-KIT on SCF/m-KIT signaling, the levels of SCF-induced phosphorylation in m-KIT were investigated in the presence of various concentrations of human recombinant s-KIT. Western blotting analysis

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Figure 1. Production of s-KIT is detected in the human epidermis and in human melanocytes. (a) Epidermal sheets from three healthy neonatal foreskins were solubilized in lysis buffer. Samples were analyzed by western blotting using m-KIT N-terminus- or b-actin-specific antibodies as detailed in Materials and Methods. The number above the figure indicates sample number. (b) Supernatant and cell lysate of cultured human melanocytes treated with or without 100 ng ml1 of PMA for 4 hours was concentrated and analyzed by western blotting using m-KIT N-terminus-specific antibody.

demonstrated that s-KIT inhibited SCF-induced phosphorylation of m-KIT in a dose-dependent manner (Figure 3b). Involvements of MMPs and TACE in s-KIT production in human melanocytes

TACE and some MMPs are involved in the production of s-KIT in mast cells (Cruz et al., 2004). To assess the role of TACE and MMPs in s-KIT production from human melanocytes, cells were treated either with an activator of TACE or MMPs (that is, APMA), a TACE inhibitor (that is, tumor necrosis factor-a protease inhibitor; TAPI-1), or inhibitors for MMPs together with PMA. Western blotting analysis with cell lysate and cultured supernatants of human melanocytes demonstrated that APMA accelerated the production of s-KIT in a dose-dependent manner, whereas expression of m-KIT decreased reciprocally (Figure 4a). In contrast, TAPI-1 and MMP inhibitor II were found to inhibit PMA-induced s-KIT production in the supernatant of cultured human melanocytes in a dose-dependent manner, whereas MMP-3 inhibitor VII had no effect on the production of s-KIT (Figure 4b), suggesting that TACE and some MMPs (except for MMP-3) mediate the production of s-KIT from human melanocytes. Production of s-KIT inhibited SCF-induced melanogenesis in human melanocytes

To examine whether s-KIT production or addition of human recombinant s-KIT inhibited SCF-induced melanogenesis in human melanocytes, human melanocytes were cultured with

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Figure 2. Time-course analysis of s-KIT production by the addition of PMA in human melanocytes. Normal human melanocytes were treated with 100 ng ml1 PMA for various incubation times. Cultured supernatant was concentrated and analyzed by western blotting using m-KIT N-terminusspecific antibody as detailed in Materials and Methods. In addition, cells were solubilized and analyzed by western blotting using m-KIT C-terminus- or b-actin-specific antibodies. Each band shows a representative result of western blotting analyses; the experiment was repeated three times with similar results. The obtained bands were analyzed with densitometer and graphs represent relative density. The values reported are means±SD. **Po0.01; ***Po0.001.

human recombinant s-KIT, APMA, or KIT-neutralizing antibody, together with human recombinant SCF for 10 days. Melanin content measured at absorbance of melanin at 405 nm revealed that induction of melanin by SCF did not occur in the presence of APMA, similar to the effect of KIT-neutralizing antibody (Figure 5), consistent with previous reports (Nishikawa et al., 1991; Okura et al., 1995; Yoshida et al., 1996). Similarly, human recombinant s-KIT was found to significantly inhibit melanin synthesis. Regulation of melanogenesis in human three-dimensional skin model

To observe the interactions between keratinocytes and melanocytes, a human three-dimensional (3D) skin model composed of human epidermal melanocytes and keratinocytes was used in this study. Models were treated either with APMA (an activator of s-KIT cleavage) or TAPI-1 (an inhibitor of s-KIT cleavage), or left untreated (control), and cultured for 14 days. Photos of the 3D skin model and melanin content demonstrated that melanogenesis was significantly suppressed by the addition of APMA, whereas TAPI-1 was found to significantly stimulate melanin synthesis (Figure 6a and b). Preferential and coordinated role of s-KIT production in the regulation of SCF/m-KIT signaling

Changes in the production of s-KIT from human melanocytes was analyzed after UVB irradiation to investigate the role of www.jidonline.org 1765

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Figure 3. Analyses of decoying activity of human recombinant s-KIT for human recombinant SCF. (a) Normal human melanocytes were treated with 1 nM of 125I-labeled SCF together with various concentrations of s-KIT for 90 minutes. Cells were solubilized and the amount of 125I-labeled SCF binding to KIT receptor on melanocytes was measured by g-counter as detailed in Materials and Methods. The nonspecific binding value (2,026) was subtracted from each value shown here. (b) Normal human melanocytes were incubated with 5 nM of human recombinant SCF together with various concentrations of human recombinant s-KIT for 10 minutes. Cells were solubilized and analyzed by western blotting using phospho-m-KIT-, m-KIT C-terminus-, or b-actin-specific antibodies as detailed in Materials and Methods. Each band shows a representative result of western blotting analyses, which were repeated three times with similar results. The obtained bands were analyzed using a densitometer. The values in the graph indicate the relative ratio of phospho-m-KIT/m-KIT. The values reported are means±SD. *Po0.05; ***Po0.001.

s-KIT in UVB-induced pigmentation, since expression of membrane-bound SCF and m-KIT has been reported to increase in epidermal keratinocyte and melanocyte, respectively, after UVB irradiation (Hachiya et al., 2001, 2004). Western blotting analyses of cultured supernatant 24 hours after various doses of UVB exposure demonstrated that production of s-KIT decreased in a dose-dependent manner, whereas m-KIT expression increased slightly (Figure 7), consistent with a previous report (Hachiya et al., 2001). Furthermore, treatment of human melanocytes with 1766 Journal of Investigative Dermatology (2008), Volume 128

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Figure 4. Activator or inhibitor of TACE and MMPs regulate the production of s-KIT in human melanocytes. (a) Normal human melanocytes were treated with 75 or 150 nM of APMA for 4 hours. Cultured supernatant was concentrated and analyzed by western blotting using m-KIT N-terminusspecific antibody as detailed in Materials and Methods. In addition, cells were solubilized and analyzed by western blotting using m-KIT C-terminus- or b-actin-specific antibodies. Each band shows a representative result of western blotting analyses, which were repeated three times with similar results. The obtained bands were analyzed using a densitometer and the graph in the figure represents relative density. The values reported are means±SD; *Po0.05. (b) Normal human melanocytes were pretreated with various concentrations of MMP inhibitor II, MMP-3 inhibitor VII, or TAPI-1 for 30 minutes before the incubation together with 100 ng ml1 of PMA for 4 hours. Cultured supernatant of the cells was concentrated and analyzed by western blotting using m-KIT N-terminus-specific antibody as detailed in Materials and Methods. Each band shows a representative result of western blotting analyses, which were repeated three times with similar results. The obtained bands were analyzed using a densitometer and the values in the graph represent relative intensity. The values reported are means±SD. **Po0.01; ***Po0.001.

human recombinant SCF for 4 hours resulted in decrease of s-KIT production (Figure 8), parallel with the decrease in m-KIT expression due to its rapid internalization and degradation after SCF binding (Blume-Jensen et al., 1991). These findings suggest a preferential and coordinated role of

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Figure 5. Production of s-KIT abolishes SCF-induced melanogenesis in human melanocytes. Normal human melanocytes were incubated with 100 nM of s-KIT, 100 nM of APMA, or 1 mg ml1 of m-KIT-neutralizing antibody, together with 10 nM of human recombinant SCF for 10 days. Cells were solubilized in 2 M NaOH. Melanin contents were measured by an absorbance meter. The values reported are means±SD. **Po0.01; ***Po0.001.

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s-KIT production in the regulation of SCF/m-KIT signaling after UVB exposure. DISCUSSION Complex coloration of the human epidermis is regulated in part by the embryonic establishment of melanocytes in the basal epithelial layer (Rosdahl and Szabo, 1978), the synthesis of melanin within the melanosome by enzymes and regulatory proteins (Imokawa and Mishima, 1982; Mishima and Imokawa, 1983), and the transfer of melanosomes from melanocytes to keratinocytes (Okazaki et al., 1976). The first two processes require a complex network of autocrine and paracrine cytokines produced in the epidermis (Halaban et al., 1988; Yada et al., 1991; Imokawa et al., 1992, 1995, 1996, 1997, 2000; Yohn et al., 1993; Schauer et al., 1994; Abdel-Malek et al., 1995; Chakraborty et al., 1996; Sirsjo et al., 1996; Wintzen and Gilchrest, 1996; Rome´ro-Gaillet et al., 1997; Funasaka et al., 1998; Hedley et al., 1998; Tada et al., 1998; Hachiya et al., 2001). These cytokines include in part basic fibroblast growth factor (FGF) (Halaban et al., 1988), endothelin-1 (Yada et al., 1991; Imokawa et al., 1992, 1996, 1997, 1995; Yohn et al., 1993; Tada et al., 1998), a-melanocyte-stimulating hormone (Schauer et al., 1994; Abdel-Malek et al., 1995; Chakraborty et al., 1996; Wintzen and Gilchrest, 1996; Funasaka et al., 1998; Hedley et al., 1998), SCF (Imokawa et al., 1996; Hachiya et al., 2001), and nitric oxide (Rome´ro-Gaillet et al., 1997). Therefore, the signaling cascades induced by cytokines are ultimately necessary to maintain cutaneous pigmentation. SCF/m-KIT signaling is regulated by increase in the expression in SCF and m-KIT in keratinocytes and melanocytes, respectively, and in phosphorylation in m-KIT after UVB irradiation, resulting in increased epidermal melanogenesis (Hachiya et al., 2001, 2004). In mast cells, hematopoietic cells, and endothelial cells, a cleaved, soluble product of KIT, s-KIT, exists (Yee et al., 1993; Broudy et al., 1994; Turner et al., 1995) that functions as a decoy receptor to regulate SCF/ m-KIT signaling (Dahlen et al., 2001). In this study, we

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Figure 6. Induction and inhibition of s-KIT production decreases and increases melanogenesis in 3D human skin models, respectively. (a) Photographs. (b) Melanin content. 3D human skin models were cultured for 14 days in the presence of 1 mM of APMA or 1 mM of TAPI-1. Cells were solubilized in 2 M NaOH followed by measurement of melanin content using an absorbance meter. The values reported are means±SD. *Po0.05; ***Po0.001.

provide evidence for the role of s-KIT in melanogenesis, which to our knowledge has not been reported previously. We demonstrate that s-KIT was detected both in the cultured supernatant of human melanocytes and the intact epidermis, and its production from m-KIT was promoted by stimulants such as PMA and APMA, an activator of TACE, and MMPs. Furthermore, s-KIT inhibited SCF-induced phosphorylation of m-KIT in human melanocytes in a dose-dependent manner, and production of s-KIT by APMA abolished SCF-induced melanogenesis. One of the most important issues addressed in this study is how s-KIT production from m-KIT abolishes SCF-induced melanogenesis in human melanocytes. It has been proposed that s-KIT binds SCF and functions as a decoy receptor to inhibit SCF/mKIT signaling (Dahlen et al., 2001). Our data indicate that binding of 125I-labeled-SCF to m-KIT in human www.jidonline.org 1767

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Figure 7. UVB irradiation increases and decreases m-KIT expression and s-KIT production, respectively. Normal human melanocytes were exposed to UVB irradiation with the indicated doses, followed by cultivation for 24 hours. Cultured supernatant was concentrated and analyzed by western blotting using m-KIT N-terminus-specific antibody as detailed in Materials and Methods. In addition, cells were solubilized and analyzed by western blotting using m-KIT C-terminus-specific antibodies or b-actin. Each band shows a representative result of western blotting analyses, which were repeated three times with similar results. The obtained bands were analyzed using a densitometer and the values in the graphs represent relative intensities. The values reported are means±SD; *Po0.05.

Figure 8. Addition of human recombinant SCF decreases the expression of m-KIT and production of s-KIT in human melanocytes. Normal human melanocytes were treated with 100 ng ml- of PMA or 10 nM of human recombinant SCF for 4 hours. Cultured supernatant was concentrated and analyzed by western blotting using m-KIT N-terminus-specific antibody as detailed in Materials and Methods. In addition, cells were solubilized and analyzed by western blotting using m-KIT C-terminus- or b-actin-specific antibodies. Each band shows a representative result of western blotting analyses, which were repeated three times with similar results. The obtained bands were analyzed using a densitometer and the values in the graphs represent relative intensities. The values reported are means±SD. *Po0.05; **Po0.01.

melanocytes was suppressed by the addition of human recombinant s-KIT in a dose-dependent manner, and was accompanied by dose-dependent interruption of SCFinduced phosphorylation in m-KIT. These results indicate that s-KIT is a potent decoy for SCF, resulting in the inhibition of SCF/m-KIT signaling in human melanocytes. However, human recombinant s-KIT was shown to only partially inhibit SCF-induced melanogenesis, whereas addition of APMA was found to abolish SCF-induced melanogenesis as effectively as KIT-neutralizing antibody, presumably due to the fact that APMA cleaves s-KIT from m-KIT, resulting in both increase in decoying s-KIT and depletion of m-KIT. The partial role of human recombinant s-KIT as a decoy for SCF may be due to lack of its glycosylation, present in the native s-KIT. Western blotting analysis revealed that human recombinant s-KIT exists as a 60-kDa protein (data not shown), whereas the fully glycosylated endogenous s-KIT exists as a 100-kDa protein in the cultured supernatant of human melanocytes and intact epidermis, consistent with a previous report indicating that endogenous s-KIT is an approximately 100-kDa polypeptide glycosylated predominantly with N-linked carbohydrate and a small amount of O-linked carbohydrate (Wypych et al., 1995). Of significance, glycosylated s-KIT expressed in baby hamster kidney cells abolishes SCF-induced cell proliferation in the murine mast cell line IC2 (Dahlen et al., 2001),

suggesting the requirement for glycosylation of s-KIT for efficient decoy function. The role of endogenous glycosylated s-KIT as a decoy in abolishing SCF-induced melanogenesis in vivo remains to be clarified. We identified a significant constitutive amount of s-KIT in the human epidermis, without any stimuli (Figure 1a). In addition, we demonstrated an increase in melanogenesis by the addition of TAPI-1, an inhibitor of TACE and MMPs, to the 3D skin model (Figure 6). These results support the role of glycosylated s-KIT in endogenously regulating cutaneous pigmentation. Expression of s-KIT from the membrane of human melanocytes may also be exogenously regulated. Previous reports demonstrated that UVB irradiation stimulates cutaneous pigmentation by inducing the expression of a variety of cytokines and their receptors, including SCF and m-KIT, in keratinocytes and melanocytes, respectively (Hachiya et al., 2001, 2004). We demonstrated that m-KIT expression was slightly increased after UVB exposure in a dose-dependent manner in human melanocytes, consistent with a previous report (Hachiya et al., 2001). Correlatively, the expression of s-KIT was decreased in a dose-dependent manner in the cultured supernatant of human melanocytes after UVB exposure. Similarly, addition of SCF to human melanocytes induced a decrease in the expression of m-KIT, consistent with a previous report demonstrating that m-KIT is phosphorylated

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and internalized rapidly after the binding of SCF to m-KIT, followed by ubiquitinization and degradation of m-KIT by proteasome (Ronnstrand, 2004). We also demonstrated an SCF induced a decrease in s-KIT in the cultured supernatant of human melanocytes, consistent with a previous report showing that addition of SCF to mast cells decreases the expression of both m-KIT and s-KIT (Cruz et al., 2004). On the other hand, it has been documented that the SCF produced by epidermal keratinocytes necessary for the maintenance of epidermal melanocytes is predominantly the membrane bound type (Longley et al., 1993; Hamann et al., 1995; Kunisada et al., 1998), and that binding of membrane-bound SCF to m-KIT results in slower internalization of m-KIT (Kapur et al., 1998). These findings indicated the preferential and coordinated role of s-KIT production in the regulation of SCF/m-KIT signaling during UVB-induced melanogenesis, by decreasing its cleavage in order to maintain SCF/m-KIT signaling. TACE is ubiquitously expressed in human tissues, including human epidermis (Black et al., 1997; Kawaguchi et al., 2004). In this study, we demonstrated that TACE and some forms of MMPs are involved in the production of s-KIT in human melanocytes. There were the following three key findings: (1) exogenous addition of PMA and APMA caused human melanocytes to produce s-KIT in a dose-dependent manner, (2) exogenous addition of a TACE inhibitor TAPI-1 and MMP inhibitor II decreased PMA-induced s-KIT production in human melanocytes in a dose-dependent manner, and (3) exogenous addition of TAPI-1 increased melanogenesis in 3D human skin model. However, the involvement of the changes in SCF/m-KIT signaling remains to be clarified in 3D human skin model after the addition of APMA and TAPI-1 to ensure the role of s-KIT production in human melanogenesis, because TACE has been reported to cleave various membrane proteins such as transforming growth factor-b and Notch, which also affect melanogenesis (Kim et al., 2004; Moriyama et al., 2006; Schouwey et al., 2007; Stavroulaki et al., 2008). Also unexplored are the specific forms of MMPs that function to produce s-KIT and the role of keratinocytes in producing s-KIT from melanocytes in a paracrine manner. Preliminary reverse transcriptase–PCR analysis data indicated that mRNA transcript expression of TACE was higher in human keratinocytes than in human melanocytes (data not shown). In conclusion, our data demonstrated that production of s-KIT was detected and increased by the addition of TACE and MMPs activators in both the cultured supernatant of human melanocytes in vitro and intact epidermis in a dosedependent manner, and that s-KIT production abolished SCF/ m-KIT signaling, resulting in complete inhibition of SCFinduced melanogenesis. To our knowledge, this information is previously unreported. These findings provide a new insight into an elaborate mechanism of SCF/m-KIT signaling in human melanocytes and suggest that production of s-KIT is involved in the regulation of human skin pigmentation. MATERIALS AND METHODS Materials Normal human epidermal melanocytes and 3D human skin models (MEL-300A) were purchased from Kurabo Corporation (Osaka,

Japan). Institutional approval was not required for experiments. Anti-m-KIT N-terminus-specific antibody was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-m-KIT C-terminusspecific antibody and recombinant SCF were provided from Immuno-Biological Laboratories Co. (Gunma, Japan). Antiphospho-m-KIT-specific antibody was purchased from Cell Signaling Technology Inc. (Danvers, MA). Anti-b-actin-specific antibody was obtained from Sigma-Aldrich Co. (St Louis, MO). Recombinant human s-KIT of over 95% purity was purchased from R&D Systems Inc. (Minneapolis, MN). A DNA sequence encoding the extracellular domain of human KIT (amino-acid residues 1–520) was expressed in Sf21 cells. Non-glycosylated recombinant s-KIT migrated as a 60-kDa protein in SDS–PAGE. Other chemicals were of reagent grade.

Cell culture Normal human epidermal melanocytes were maintained in Medium 254 (Kurabo Corporation) supplemented with 5 mg ml1 of insulin, 5 mg ml1 of transferrin, 3 ng ml1 of human recombinant FGF, 0.18 mg ml1 of hydrocortisone, 3 mg ml1 of heparin, 10 ng ml1 of PMA, 0.2% (v/v) of bovine putative extract, and 0.5% (v/v) of fetal bovine serum at 37 1C with 5% CO2, as described previously (Yada et al., 1991).

Western blotting To investigate the protein expression levels of m-KIT and/or s-KIT cleavage product in human cultured melanocytes and cultured supernatant, respectively, subconfluent melanocytes in a dish of 10 cm diameter were cultured in the conditioned medium (Medium 254 supplemented with 5 mg ml1 of insulin, 5 mg ml1 of transferrin, 3 ng ml1 of human recombinant FGF, 0.18 mg ml1 of hydrocortisone, 3 mg ml1 of heparin, 0.2% (v/v) of bovine putative extract, and 0.5% (v/v) of fetal bovine serum) for 3 days before the addition of stimuli. Then used media were replaced by fresh conditioned medium (Medium 254 supplemented with 5 mg ml1 of insulin, 3 ng ml1 of human recombinant FGF, and 3 mg ml1 of heparin) and 100 ng ml1 of PMA, 10 nM of human recombinant SCF, APMA (75–150 nM) (EMD Biosciences Inc., San Diego, CA) , MMP inhibitor II (2–20 mM) (EMD Biosciences Inc.), MMP III inhibitor VII (2–20 mM) (EMD Biosciences Inc.), or TAPI-1 (2–20 mM) (EMD Biosciences, Inc.) was added to the culture. In another experiment, human melanocytes were exposed to UVB irradiation by UVB lamps (Toshiba SE lamps for UVB with a peak of emission near 312 nm). To examine the expression levels of m-KIT, human melanocyte-derived protein (5 mg) solubilized in cell lysis buffer (Cell Signaling Technology) with 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich Co.) was separated on 7.5% SDS gels (Bio-Rad Laboratories, Hercules, CA). To detect the s-KIT cleaved from melanocyte surface, each cultured supernatant was filtered with a 0.45-mm membrane (Millipore Co., Billerica, MA) and quantified. The filtered supernatant was concentrated over 40-fold using centricon plus 20. The reduced supernatant was then quantified. On the basis of this quantification, supernatants were then subsequently diluted with distilled water in order to attain a final concentration of 40-fold, thus normalizing all samples. Twelve microliters of concentrated supernatant were separated on 7.5% SDS gel. They were then transferred to SequiBlot PVDF membrane (Bio-Rad Laboratories) and incubated with a purified polyclonal rabbit antibody specific for the C-terminus of www.jidonline.org 1769

S Kasamatsu et al. Role of the Soluble Form of KIT in Melanocytes

m-KIT (0.1 mg ml1) or for the N-terminus of m-KIT (0.2 mg ml1), followed by incubation with secondary antibody corresponding to rabbit IgG (GE Healthcare UK Ltd, Buckinghamshire, UK). Subsequent visualization of antibody recognition was performed using Enhanced ChemiLuminescence Plus (GE Healthcare UK Ltd) according to the manufacturer’s instructions. Subconfluent melanocytes in six-well-plates were cultured in the conditioned medium (Medium 254 supplemented with 5 mg ml1 of insulin, 5 mg ml1 of transferrin, 3 ng ml1 of human recombinant FGF, 0.18 mg ml1 of hydrocortisone, 3 mg ml1 of heparin, 0.2% (v/v) of bovine putative extract, and 0.5% (v/v) of fetal bovine serum) for 3 days before addition of stimuli to analyze the phosphorylation level of m-KIT. The media were then changed to fresh conditioned medium and 10 nM of human recombinant SCF and/or various concentrations of s-KIT were added to the media. Following 10 minutes of stimuli, the plates were washed twice with ice-cold phosphate-buffered saline (PBS) and cells were solubilized in cell lysis buffer with 1 mM of phenymethylsulfonyl fluoride. The samples were separated on 7.5% SDS gels, transferred to Sequi-Blot PVDF membrane, and incubated with a purified polyclonal rabbit antibody specific for phospho-m-KIT (1,000-fold dilution). Subsequent visualization was performed as described above. To examine the protein expression levels of m-KIT and s-KIT cleavage in human epidermis in vivo, neonatal foreskin was obtained from routine circumcision (Christ Hospital, Cincinnati, OH) and incubated at 80 1C for 1 day. Immediately after incubation in H2O for 2 minutes at 60 1C, tissue was kept on ice for 10 minutes in order to separate epidermal sheet from the underlying dermis. The epidermal samples were then lysed using a cell lysis buffer supplemented with 1 mM of phenylmethylsulfonyl fluoride and 170 mg of solubilized protein were separated on 7.5% SDS gels to detect the expression of m-KIT and s-KIT. Subsequent procedures were performed as described above. In each experiment above, the amount of protein loaded was normalized against a control protein, b-actin, with a mAb specific for b-actin (Sigma-Aldrich Co.).

Radiolabeling and ligand-binding assay The SCF binding assay was performed as described elsewhere (Lev et al., 1992). Human recombinant SCF was labeled with 125I (GE Healthcare UK Ltd) using IODO-GEN iodination reagent (Pierce Biotechnology Inc. Rockford, IL). For the binding assay, subconfluent melanocytes in 24-well plates were washed with PBS and were then incubated with 1 nM of 125I-labeled SCF and a variety of concentrations of s-KIT in 300 ml of binding buffer (RPMI 1640 medium containing 0.05% BSA) for 90 minutes at 37 1C. The plates were rinsed five times with ice-cold PBS and melanocytes were solubilized in 500 ml of 2 M NaOH; radioactivity was counted in a g-counter (Canberra Packard GmbH, Schwadorf, Austria). Nonspecific binding was determined by parallel binding experiments in the presence of 100-fold excess of unlabeled SCF.

Measurement of melanin content in human cultured melanocytes To measure the amount of melanin content in human melanocytes, 5  104 melanocytes were cultured in the conditioned medium (Medium 254 supplemented with 5 mg ml1 of insulin, 5 mg ml1 of transferrin, 3 ng ml1 of human recombinant FGF, 0.18 mg ml1 of 1770 Journal of Investigative Dermatology (2008), Volume 128

hydrocortisone, 3 mg ml1 of heparin, 0.2% (v/v) of bovine putative extract, and 0.5% (v/v) of fetal bovine serum) in six-well-plates and 100 nM of human recombinant s-KIT, 100 nM of APMA, or 1 mg ml1 of KIT-neutralizing antibody (K44.2; Lab Vision Co., Fremont, CA) was added to the culture together with 10 nM of human recombinant SCF. After culturing for 10 days, the wells were washed three times with PBS and cells were solubilized in 200 ml of 2 M NaOH. Melanin content in human cultured melanocytes was measured by an absorbance meter (Microplate Reader Model 550; Bio-Rad Laboratories) using a 405-nm filter.

Measurement of melanin content in 3D human skin models According to the manufacturer’s instructions, 3D human skin models were maintained in EPI-100LLMM medium (Kurabo Corporation) at 37 1C with 5% CO2. Skin models were incubated with 1 mM APMA or 1 mM TAPI-1 for 14 days. Photographs of skin models were taken when cells were harvested. Cells were washed three times with PBS, 5% (v/v) of trichloroacetic acid, and diethyl ether mixed with three times volume of ethanol in turn. Then, cells were washed once with diethyl ether and incubated at 50 1C for 2 hours until dry. Cells were solubilized in 200 ml of 2 M NaOH. Melanin content in 3D human skin model was measured by an absorbance meter using a 405-nm filter, as described above.

Statistics The level of significance of the difference was calculated by the Student’s t-test. The differences in the mean or raw values among treatment groups were considered significant when Po0.05. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank Y. Shimotoyodome for technical assistance.

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