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Differentiation of Murine Melanocyte Precursors Induced by 1,25-Dihydroxyvitamin D3 Is Associated with the Stimulation of Endothelin B Receptor Expression
Differentiation of Murine Melanocyte Precursors Induced by 1,25-Dihydroxyvitamin D3 Is Associated with the Stimulation of Endothelin B Receptor Expression Hidenori Watabe, Yoshinao Soma, Yoko Kawa, Masaru Ito, Shiho Ooka, Kayoko Ohsumi, Takako Baba, Tamihiro Kawakami, Eri Hosaka, Satoko Kimura, and Masako Mizoguchi Department of Dermatology, St Marianna University School of Medicine, Kawasaki, Japan
treatment with 1,25-dihydroxyvitamin D3. The induction of endothelin B receptor by 1,25-dihydroxyvitamin D3 was also demonstrated in neural crest cell primary cultures, but not in mature melanocytes. The expression of microphthalmiaassociated transcription factor was induced in NCC-/melb4 cells treated with 1,25-dihydroxyvitamin D3 and endothelin 3, but not by 1,25dihydroxyvitamin D3 alone, suggesting that endothelin 3 may stimulate the expression of the microphthalmia-associated transcription factor gene after binding to the endothelin B receptor induced by 1,25-dihydroxyvitamin D3. These ®ndings suggest a regulatory role for vitamin D3 in melanocyte development and melanogenesis, and may also explain the working mechanism of vitamin D3 in the treatment of vitiligo. Key words: endothelin 3/melanogenesis/microphthalmia-associated transcription factor/ vitamin D receptor/vitamin D3. J Invest Dermatol 118:583±589, 2002
The effects of 1,25-dihydroxyvitamin D3 on the differentiation of immature melanocyte precursors were studied. The NCC-/melb4 cell line is an immature melanocyte cell line established from mouse neural crest cells. 1,25-dihydroxyvitamin D3 inhibited the growth of NCC-/melb4 cells at concentrations higher than 10±8 M. That growth inhibition was accompanied by the induction of tyrosinase and a change in L-3,4-dihydroxyphenylalanine reactivity from negative to positive. Electron microscopy demonstrated that melanosomes were in more advanced stages after 1,25-dihydroxyvitamin D3 treatment. In primary cultures of murine neural crest cells, L-3,4dihydroxyphenylalanine-positive cells were increased after 1,25-dihydroxyvitamin D3 treatment. These ®ndings indicate that 1,25-dihydroxyvitamin D3 stimulates the differentiation of immature melanocyte precursors. Moreover, immunostaining and reverse transcription±polymerase chain reaction analysis revealed that endothelin B receptor expression was induced in NCC-/melb4 cells following
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(VDR) (Haussler, 1986), which is a member of the superfamily of nuclear steroid/thyroid receptors. After binding 1,25(OH)2D3, the receptors form homodimers and heterodimers with other members of the superfamily and act as transcription factors for a variety of genes (Carlberg, 1993). This is referred to as the classic genomic effect of 1,25(OH)2D3 because it involves gene transcription and new protein synthesis. Vitamin D3 stimulates the differentiation and inhibits the proliferation of many different types of tumor cells, including a variety of melanoma cell lines. Murine B16 melanoma cells treated with vitamin D3 exhibit an increase in tyrosinase activity and melanogenesis (Oikawa and Nakayasu, 1974). A signi®cant growth inhibition was noted in human melanoma cell lines treated with vitamin D3 (Evans et al, 1996). There has been a report showing that vitamin D3 did not affect melanization of cultured human melanocytes (Mansur et al, 1988), but in apparent contrast, Tomita et al (1988) showed that vitamin D3 increased the tyrosinase content of cultured human melanocytes. Topical application of vitamin D3 to the epidermis of mice increases the number of L-3,4dihydroxyphenylalanine (DOPA)-positive melanocytes (AbdelMalek et al, 1988). These ®ndings suggest a possible role for vitamin D3 in modulating melanogenesis, but little is known
he active metabolite of vitamin D, namely 1,25dihydroxyvitamin D3 (1,25(OH)2D3), is known for its regulatory role in calcium homeostasis. The hormone, derived either from 7-dehydrocholesterol by the action of ultraviolet light on the skin or from the diet, facilitates the absorption of calcium from the intestine, its mobilization from bone, and its resorption in the kidney (BurgosTrinidad et al, 1990; De Luca et al, 1990; Studzinski et al, 1993). Apart from its role in calcium uptake, 1,25(OH)2D3 is widely acknowledged to be an important regulator of cell growth and differentiation (Lowe et al, 1992; Darwish and De Luca, 1993; Nemere et al, 1993). The biologic responses elicited by 1,25(OH)2D3 have primarily been attributed to the activation of the nuclear vitamin D receptor Manuscript received May 15, 2002; accepted for publication June 20, 2002 Reprint requests to: Yoshinao Soma, MD, Department of Dermatology, St Marianna University School of Medicine, 2-16-1, Sugao, Miyamae-ku, Kawasaki, 216-8511, Japan. Email: [email protected] Abbreviations: EDN, endothelin; EDNRB, endothelin B receptor; NCC, neural crest cells; SCF, stem cell factor; 1,25(OH)2D3, 1,25dihydroxyvitamin D3; VDR, vitamin D receptor. 0022-202X/02/$15.00
´ Copyright # 2002 by The Society for Investigative Dermatology, Inc. 583
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concerning the effect of 1,25(OH)2D3 on the differentiation of melanocyte precursors or cultured neural crest cells (NCC). We have established an immortal cell population of NCC from WB Sl/+ or +/+ mice, and have designated it as NCC-S4.1 (Kawa et al, 2000). From NCC-S4.1 cells, we obtained a cloned cell line named NCC-melb4 by a single-cell cloning method (Kawa et al, 2000). NCC-melb4 cells are immature melanocyte precursors, as they are positive for tyrosinase-related protein-1 and -2 and KIT, but are negative for tyrosinase, endothelin B receptor (EDNRB), and DOPA reaction (Watabe et al, 2002). They are immortal and stable when maintained in a culture medium supplemented with stem cell factor (SCF). Their growth is not dependent on endothelin 3 (EDN3), probably because of their lack of EDNRB expression. We have also obtained a cloned cell line named NCCmelan5 from C57BL/6 mice (Ooka et al, 2001). NCC-melan5 cells require SCF and EDN3 in the culture medium. They appear to be mature melanocytes, as they are positive for tyrosinase, tyrosinaserelated protein-1 and -2, KIT, EDNRB, and DOPA reactions. Using these cell lines and mouse NCC in a primary culture system, the effects of 1,25(OH)2D3 on the differentiation of the cells and their expression of EDNRB were investigated.
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95°C for 60 s (EDNRB) or at 94°C for 10 s (VDR), annealing at 60°C for 30 s (tyrosinase and MITF), at 60°C for 60 s (EDNRB) or at 58°C for 60 s (VDR), polymerization at 72°C for 60 s (tyrosinase and MITF), at 72°C for 120 s (EDNRB) or at 72°C for 30 s (VDR). The primer sequences used were as follows: Tyrosinase sense primer 5¢-CTATGTCATCCCCACAGGCAC-3¢ Tyrosinase anti-sense primer 5¢-TTACCTGCCAGGAGGAGAAGA-3¢ MITF sense primer 5¢-AGTCACTACCAGGTGCAGAC-3¢ MITF anti-sense primer 5¢-CTTGCTTCAGACTCTGTGGG-3¢ EDNRB sense primer 5¢-CGAGCTGTTGCTTCTTGGAGTAG-3¢ EDNRB anti-sense primer 5¢-AACGGAAGTTGTCATATCCGTGAT-3¢ VDR sense primer 5¢-AGATGACCCTTCTGTGACCC-3¢ VDR anti-sense primer 5¢-AGCTTCTTCAGTCCCACCTG-3¢ b-actin primers were from Promega, Inc. (Madison, WI). PCR products were resolved in 2% agarose gels containing ethidium bromide in parallel with DNA molecular weight markers.
Mice C57BL/6 mice purchased from Japan SLC Co., Ltd. (Hamamatsu, Japan) were mated, and the embryos from those matings were used at 9.5 d postcoitum (E9.5).
Western blot analysis Cells were harvested by scraping, collected by centrifugation, and were then lyzed in cell lysis buffer (1% Triton X-100, 10 mM Tris±HCl, pH 7.4). Cell lysates were separated on 10% sodium dodecyl sulfate polyacrylamide gels. After electrophoresis, proteins on the gel were electroblotted on to nitrocellulose ®lters that were then incubated for 2 h in Tris-buffered saline (100 mM NaCl, 50 mM Tris±HCl, pH 7.2) containing 2.5 mg per ml nonfat powdered milk (Tris-buffered saline milk). The ®lters were then incubated overnight in the presence of the tyrosinase antibody (aPEP7, 1 : 500) and was then reacted with alkaline Anti-phosphatase-labeled anti-rabbit IgG (DAKO) for 90 min. The ®lters were washed in Tris-buffered saline milk and the antigens were detected with 5-bromo-4-chloro-3-indolyl phosphate (Sigma) and nitroblue tetrazodium (Sigma).
Cells and culture conditions NCC-melb4 cells were established using a single cell cloning method from an immortal cell population (NCCS4.1) obtained from NCC derived from WB Sl/+ or +/+ mice (Kawa et al, 2000). NCC-melan5 cells were established from NCC of C57BL/6 mice (Ooka et al, 2001). NCC-melb4 cells were grown in Eagle's modi®ed essential medium supplemented with 5% fetal bovine serum and 50 ng per ml recombinant mouse SCF (kindly supplied by Kirin Brewery, Tokyo, Japan). NCC-melan5 cells were grown in Eagle's modi®ed essential medium containing 15% fetal bovine serum, 50 ng per ml recombinant mouse SCF, and 100 nM EDN3 (Peptide Institute Inc, Osaka, Japan). 1,25(OH)2D3 (BIOMOL Research Laboratories, Inc, Plymouth Meeting, PA) was dissolved in ethanol at a concentration of 10±4 M as a stock solution, and then was added to the culture medium at a ®nal concentration of 10±7 M, unless indicated otherwise.
Statistical analysis
MATERIALS AND METHODS
Proliferation assay Cells were plated at 2000 cells per well in 96-well plates. On the next day various concentrations of 1,25(OH)2D3 were added to the culture medium (day 0). On day 5, the cells were incubated with alamar blue dye (Trek Diagnostic Systems, Inc, Westlake, OH) for 4 h and the ¯uorescence was measured using a Fluoroskan II microplate reader (Labsystems, Helsinki, Finland) Immunohistochemistry The antibody to tyrosinase (aPEP7, which recognizes the carboxy terminus of tyrosinase, Jimenez et al, 1991) were kindly supplied by Dr Hearing (NIH, Bethesda, MD). Cells ®xed with 95% ethanol at room temperature were treated with antibodies against tyrosinase, EDNRB (IBL, Maebashi, Japan) or VDR (Diagnostic System Laboratories, Webster, TX) as described previously (Kawa et al, 2000; Ooka et al, 2001; Watabe et al, 2002). They were then reacted with alkaline phosphatase-labeled anti-rat IgG (Southern Biotechnology Associates, Birmingham, LA) or rabbit IgG (DAKO, Carpinteria, CA) and developed using the New Fuchsin Substrate System (DAKO). DOPA reaction and electron microscopy Cells were ®xed with 2% paraformaldehyde at room temperature and were then treated with 0.1% DOPA (Sigma, St Louis, MO) in 0.1 M phosphate buffer (pH 7.2) for 5 h at 37°C. Nuclei were stained by Nuclear fast red (Merck, Darmstadt, Germany). Electron microscopy and electron microscopic DOPA staining were performed as described elsewhere (Kawa et al, 2000). RNA extraction and reverse transcription±polymerase chain reaction (reverse transcription±PCR) analysis Total RNA was extracted from the cells using an RNA isolation kit (QIAGEN, Hilden, Germany) as described by the manufacturer. cDNA was prepared from 10 mg total RNA using MMLV Reverse Transcriptase (Gibco/BRL, Gaithersburg, MD). PCR reactions consisted of 34 cycles for tyrosinase and microphthalmia-associated transcription factor (MITF), 35 cycles for EDNRB or 40 cycles for VDR. PCR reactions were initiated with a ``cold start''; denaturation at 98°C for 10 s (tyrosinase and MITF), at
NCC culture The NCC culture method was that described by Ito and Takeuchi (1984). Trunk regions posterior to the forelimb buds were dissected from E9.5 embryos of C57BL/6 mice using tungsten needles. The trunks were individually treated with 1% trypsin (Difco, Detroit, MI) in Tyrode's solution for 20 min at 4°C. Trypsinization was terminated by washing with Tyrode's solution containing 10% fetal bovine serum. The tissues were gently pipetted with small pore Pasteur pipettes to separate the neural tubes from other components of the trunks. Neural tubes were explanted individually in 12-well plates with Eagle's modi®ed essential medium containing 15% fetal bovine serum and 50 ng per ml recombinant mouse SCF. The explants were ®xed at 13 d in culture and were then subjected to DOPA reaction or immunohistochemistry. Student's t test was used for statistical analysis.
RESULTS Inhibitory effects of 1,25(OH)2D3 on the proliferation of NCC-melb4 cells We studied whether 1,25(OH)2D3 affected the proliferation of NCC-melb4 cells (Fig 1). Cellular proliferation was inhibited by 1,25(OH)2D3 in a concentration-dependent manner and was markedly inhibited at concentrations higher than 10±8 M. Equivalent amounts of vehicle (ethanol) alone had no effects on the cell growth (data not shown). Differentiation of NCC-melb4 cells induced by 1,25(OH)2D3 As NCC-melb4 cells are immature melanocytes, we considered that the growth inhibition elicited by 1,25(OH)2D3 might be associated with their differentiation. We then examined the effects of 1,25(OH)2D3 on the expression of tyrosinase and DOPA reactivity as markers of differentiation. NCC-melb4 cells were initially negative for tyrosinase and DOPA reactivity (Fig 2a,c). Presumably because NCC-melb4 was a cloned cell line, all cells were negative for both reaction without exception. After 72 h of treatment with 1,25(OH)2D3, some but not all of them became tyrosinase positive and DOPA positive (Fig 2b,d). The induction of tyrosinase by 1,25(OH)2D3 was also demonstrated by western blot analysis (Fig 3a) and by reverse transcription±PCR analysis (Fig 3b). These results suggest that 1,25(OH)2D3 stimulates the differentiation of immature melanocyte precursors. 1,25(OH)2D3 induces EDNRB expression Next we examined whether 1,25(OH)2D3 affects EDNRB expression in NCC-melb4 cells. The cells were originally negative for EDNRB
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Figure 3. Enhanced expression of tyrosinase after 1,25(OH)2D3 treatment. Tyrosinase expression in NCC-melb4 cells before and after the treatment with 10±7 M 1,25(OH)2D3 for 72 h was determined by western blotting (a) and by reverse transcription±PCR analysis (b) as described in Materials and Methods. (b) Lanes 1 and 2: b-actin; lanes 3 and 4: tyrosinase.
Figure 1. 1,25(OH)2D3 inhibits cell proliferation in a concentration-dependent manner. NCC-melb4 cells were plated at 2000 cells per well in 96-well plates and were incubated with various concentrations of 1,25(OH)2D3. After 5 d, cells were incubated with alamar blue dye and the ¯uorescence was measured on a microplate reader. **p < 0.01.
in Fig 4(a,b), 1,25(OH)2D3 induced the expression of EDNRB mRNA in NCC-melb4 cells at 10±7 M and 10±8 M, but not at 10±9 M. We then investigated whether 1,25(OH)2D3 alters EDNRB expression in NCC-melan5 cells, which are EDNRBpositive mature melanocytes (Ooka et al, 2001). As shown in Fig 4(c), 1,25(OH)2D3 had no effect on the expression of EDNRB mRNA in NCC-melan5 cells, although NCC-melb4 cells and NCC-melan5 cells expressed VDR mRNA, as demonstrated by reverse transcription±PCR analysis (Fig 5). Differentiation of NCC-melb4 cells after 1,25(OH)2D3 treatment at the ultrastructural level Electron microscopy and ultrastructural DOPA staining are shown in Fig 6. Before treatment with 1,25(OH)2D3, NCC-melb4 cells contained stage I melanosomes only, none being in more advanced stages, and they were DOPA negative. After 72 h of treatment with 1,25(OH)2D3, stage III±IV melanosomes became apparent, and DOPA staining was positive in the Golgi areas and in melanosomes. The combination of 1,25(OH)2D3 and EDN3 induces MITF expression NCC-melb4 cells were incubated with 1,25(OH)2D3 alone or in the presence of EDN3 for 72 h, and were then tested for MITF expression by reverse transcription± PCR analysis (Fig 7). The combination of 1,25(OH)2D3 and EDN3 induced MITF mRNA expression, whereas 1,25(OH)2D3 alone did not. This suggests a possible role for EDNRB signaling in the induction of MITF, and that EDNRB induced by 1,25(OH)2D3 may effectively transduce signals that stimulate MITF expression.
Figure 2. Effects of 1,25(OH)2D3 on tyrosinase expression, DOPA reaction and EDNRB expression. NCC-melb4 cells before (a,c,e) and after (b,d,f) treatment with 10±7 M 1,25(OH)2D3 for 72 h were immunohistochemically stained for tyrosinase (a,b) or EDNRB (e,f), or were tested for DOPA reaction (c,d). Scale bar = 10 mm.
(Fig 2e), but after 72 h of treatment with 10±7 M 1,25(OH)2D3, some but not all of them became EDNRB positive (Fig 2f). To study the induction of EDNRB by 1,25(OH)2D3 at the molecular level, reverse transcription±PCR analysis was performed. As shown
EDNRB-positive cells and DOPA-positive cells increases in NCC cultures after 1,25(OH)2D3 treatment We investigated the effect of 1,25(OH)2D3 on the NCC primary culture system. Explants from C57BL/6 mice embryos contained VDR-positive cells in NCC around the periphery of the epithelial sheets (data not shown). Before treatment with 1,25(OH)2D3, the explants were negative for EDNRB, but after 1,25(OH)2D3 treatment, EDNRBpositive cells appeared in NCC around the periphery of the epithelial sheets (Fig 8). As shown in Fig 9, an increase in DOPApositive cells was seen following treatment of the NCC cultures with 1,25(OH)2D3. DISCUSSION Various factors, including SCF, EDN3, 12-O-tetradecanoylphorbol-13-acetate, and cholera toxin, have been shown to stimulate the differentiation of melanocyte precursors (Murphy
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Figure 5. Reverse transcription±PCR analysis of VDR mRNA expression in NCC-melb4 cells and in NCC-melan5 cells. Total RNA was reverse-transcribed followed by 40 cycles PCR of cDNA using the primers described in Materials and Methods. Lane 1: b-actin of NCC-melb4 cells; lane 2: b-actin of NCC-melan5 cells; lane 3: VDR of NCC-melb4 cells; lane 4: VDR of NCC-melan5 cells.
Figure 4. EDNRB expression is induced by 1,25(OH)2D3 in NCC-melb4 cells but not in NCC-melan5 cells. (a) NCC-melb4 cells were incubated with or without 10±7 M 1,25(OH)2D3 for 72 h. Total RNA was extracted and analyzed by reverse transcription±PCR. Lanes 1 and 2: b-actin; lanes 3 and 4: EDNRB. (b) NCC-melb4 cells were incubated with various concentrations of 1,25(OH)2D3 for 72 h. Total RNA was extracted and analyzed by reverse transcription±PCR. Lane 1: 10±7 M; lane 2: 10±8 M; lane 3: 10±9 M; lane 4: size marker. (c) NCC-melan5 cells were incubated with or without 10±7 M 1,25(OH)2D3 for 72 h. Total RNA was extracted and analyzed by reverse transcription±PCR. Lanes 1 and 2: b-actin; lanes 3 and 4: EDNRB.
et al, 1992; Ito et al, 1993; Ono et al, 1998), but the effect of 1,25(OH)2D3 on the differentiation of melanocyte precursors and on NCC cultures has not yet been established. VDR expression has been detected in several cancer cell lines, including colon carcinoma, breast carcinoma, and malignant melanoma (Colston et al, 1981; Frampton et al, 1982), and in human melanocytes in vivo (Milde et al, 1991). In this study, we show that VDR mRNA is expressed in two murine NCC lines, i.e., NCC-melb4 and NCCmelan5, and that neural crest explants from C57BL/6 mice embryos contain VDR-positive cells in NCC around the periphery of the epithelial sheets, which suggests a possible role for vitamin D in regulating the proliferation and differentiation of melanocyte precursors. In this study, we show that 1,25(OH)2D3, which is the active metabolite of vitamin D, inhibits the growth of NCC-melb4 cells. That growth inhibition is accompanied by the induction of tyrosinase and a change in DOPA reactivity from negative to positive. Electron microscopy demonstrates the presence of melanosomes at more advanced stages in 1,25(OH)2D3-treated cells, compared with untreated cells. In primary NCC cultures, DOPA-positive cells are increased after 1,25(OH)2D3 treatment. These ®ndings indicate that 1,25(OH)2D3 may stimulate the differentiation of immature melanocyte precursors.
Endothelins (EDN1, EDN2, EDN3) are paracrine signal peptides (21 amino acid residues in length) that bind to at least two subtypes of G-protein-coupled heptahelical receptors (EDNRA and EDNRB). Those two receptors share high sequence homology (Elshourbagy et al, 1993), but can be distinguished by their speci®city; however, EDNRA binds EDN1 and EDN2 with higher af®nity, all three EDN are bound equally well by EDNRB (Sakurai et al, 1992). EDN were shown to be bound by normal human melanocytes that respond with increased proliferation and pigmentation (Yada et al, 1991; Imokawa et al, 1992, 1995, 1997). EDN3 also promotes the survival and proliferation of melanocyte precursors (Lahav et al, 1996, 1998; Ono et al, 1998). The expression of EDNRB is directly regulated by tumor necrosis factor a in human endothelial cells (Smith et al, 1998), but the regulation of EDNRB expression in melanocyte precursors remains unknown. Immunostaining and reverse transcription±PCR analysis shown in this study reveal the induction of EDNRB by 1,25(OH)2D3 in NCC-melb4 cells and also in NCC primary cultures. These ®ndings indicate that 1,25(OH)2D3 acts to promote the expression of EDNRB in immature melanocyte precursors, and they also suggest a possible role for 1,25(OH)2D3 in modulating melanocyte development in vivo. To the best of our knowledge, this is the ®rst study reporting the effect of vitamin D3 on EDNRB expression, and moreover, no other factors have been shown to stimulate EDNRB expression in melanocytes or in immature melanocyte precursors. Recently, we showed that all-trans retinoic acid induces the differentiation of NCC-melb4 cells, but that it did not induce EDNRB (Watabe et al, 2002). We have also studied whether other factors, such as SCF, EDN, 12-O-tetradecanoyl-phorbol-13-acetate, or cholera toxin, induce EDNRB on NCC-melb4 cells and found that none of them did (data not shown). Therefore, the induction of EDNRB on melanocyte precursors may be a speci®c function of 1,25(OH)2D3. As EDNRB is one of the marker molecules for mature melanocytes, vitamin D3 may be an essential factor in melanocyte development. Reverse transcription±PCR analysis showed that 1,25(OH)2D3 had no effect on the expression of EDNRB in NCC-melan5 cells (EDNRB-positive mature melanocytes), suggesting that 1,25(OH)2D3 may stimulate EDNRB expression by immature melanocyte precursors such as NCC-melb4 cells but not in mature melanocytes such as NCCmelan5 cells. MITF is the human homolog of the mouse microphthalmia (mi) gene product, which is a novel transcription factor of the basic± helix±loop±helix (bHLH)±leucine zipper protein family (Hodgkinson et al, 1993; Hughes et al, 1993; Tachibana et al, 1994). MITF is able to direct melanocyte-speci®c transcription of the tyrosinase and tyrosinase-related protein-1 genes (Yasumoto et al, 1994, 1997), and is thought to be involved in regulating the differentiation and maintenance of melanocytes (Tachibana, 1997, 2000). It has been suggested that EDN may regulate MITF function at the protein and promoter levels (Tachibana, 2000). This study demonstrates that 1,25(OH)2D3 combined with EDN3 induces MITF expression in NCC-melb4 cells, whereas 1,25(OH)2D3 alone does not. Thus, EDN3 may induce expression
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Figure 6. Electron microscopy and ultrastructural DOPA staining reveal the development of melanocyte precursors with 1,25(OH)2D3. Electron microscopy (a,c) and electron microscopic DOPA staining (b,d) of NCC-melb4 cells were made before (a,b) and after (c,d) 72 h of treatment with 10±7 M 1,25(OH)2D3. Before the treatment, NCC-melb4 cells contained only stage I melanosomes (small arrow) and were DOPA negative. After the treatment, stage III±IV melanosomes (large arrows) were seen, and both Golgi areas and melanosomes were DOPA-positive. Scale bar = 1 mm.
Figure 7. Induction of MITF mRNA by combined treatment with 1,25(OH)2D3 and EDN3. Reverse transcription±PCR analysis of MITF mRNA from NCC-melb4 cells treated with or without 10±7 M 1,25(OH)2D3 and 100 nM EDN3 for 72 h. Lane 1: no addition; lane 2: 1,25(OH)2D3; lane 3: 1,25(OH)2D3 + EDN3; lane 4: size marker.
of the MITF gene in the presence of 1,25(OH)2D3. It also appears that EDNRB induced by 1,25(OH)2D3 functions as the receptor for EDN3, being capable of signal transduction that effectively stimulates MITF expression. There have been several reports of hyperpigmentation resulting from the combined use of topical calcipotriol (a vitamin D analog) and phototherapy in patients with psoriasis or vitiligo vulgaris (Kokelj et al, 1995; GlaÈser et al, 1998; Parsad et al, 1998). Recent clinical trials demonstrated the ef®cacy of topical calcipotriol combined with psoralen and ultraviolet A in treating vitiligo (Yalcin et al, 2001; Ameen et al, 2001; Ermis et al, 2001); however, the working mechanism of vitamin D in that treatment regimen remains unknown. In the treatment of vitiligo, repigmentation is usually perifollicular (Kovacs, 1998), indicating that melanin is
Figure 8. 1,25(OH)2D3 stimulates the expression of EDNRB in primary NCC cultures. Neural tubes of C57BL/6 mice were explanted and cultured with or without 10±7 M 1,25(OH)2D3. The explants were ®xed at 13 d in culture and were subjected to immunohistochemical staining for EDNRB. Note that numerous EDNRB-positive cells (reddish staining) are seen in 1,25(OH)2D3treated cells (b), but not in nontreated cells (a).
produced by the melanocytes in hair follicles. In our study, 1,25(OH)2D3 induces EDNRB expression in immature melanocytes such as NCC-melb4 cells, but not in mature melanocytes such as NCC-melan5 cells. A melanocyte stem cell-like sub-
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Figure 9. 1,25(OH)2D3 increases DOPA-positive cells in primary NCC cultures. Neural tubes of C57BL/6 mice were explanted and cultured with or without 10±7 M 1,25(OH)2D3. The explants were ®xed at 13 d in culture and were subjected to the DOPA reaction. Note that numerous DOPA-positive cells are seen in 1,25(OH)2D3-treated cells (b), but not in nontreated cells (a).
population is thought to be present in the bulge region of hair follicles (Botchkareva et al, 2001). Taken together, we consider that vitamin D may act to induce immature melanocytes in hair follicles to produce melanin by stimulating their differentiation and their expression of EDNRB. Thus, the effects of 1,25(OH)2D3 presented in this study provide some important clues to understand the roles of vitamin D3 in melanocyte development and melanogenesis. It appears that hyperpigmentation is induced not by a calcipotriol monotherapy, but by a combination of phototherapy plus calcipotriol, as most patients with psoriasis who are treated with calcipotriol do not show any increase in skin pigmentation. Then it is considered that there must be some additional effects of the phototherapy on the melanocyte signaling that are independent of the vitamin D effect. EDN secreted by ultraviolet B-exposed keratinocytes are one of the candidates that cause ultravioletinduced hyperpigmentation (Imokawa et al, 1995, 1997). Further studies using human melanocytes and melanocyte precursors are necessary, as the results with murine cells may not be directly applied to human cells. We would like to sincerely thank Dr Hearing (NIH, Bethesda, MD) for providing us with the antibodies for tyrosinase. We would also like to sincerely thank the Kirin Brewery (Tokyo, Japan) for providing us with SCF. This work was supported by grants from the Scienti®c Research Fund of the Ministry of Education, Science, Sports and Culture, Japan (Grant-in Aid for Scienti®c Research, no. 12670843).
REFERENCES Abdel-Malek ZA, Ross R, Trinkle L, et al: Hormonal effects of vitamin D3 on epidermal melanocytes. J Cell Physiol 136:273±280, 1988 Ameen M, Exarchou V, Chu AC: Topical calcipotriol as monotherapy and in combination with psoralen plus ultraviolet A in the treatment of vitiligo. Br J Dermatol 145:476±479, 2001 Botchkareva NV, Khlgatian M, Longley BJ, et al: SCF/c-kit signaling is required for cyclic regeneration of the hair pigmentation unit. FASEB J 15:645±658, 2001 Burgos-Trinidad M, De Brown AJ, Luca HF: A rapid assay for 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D 24-hydroxylase. Anal Biochem 190:102±107, 1990 Carlberg C: RXR-independent action of the receptors for thyroid hormone, retinoid acid and vitamin D on inverted palindromes. Biochem Biophys Res Commun 195:1345±1353, 1993 Colston K, Colston MJ, Feldman D: 1,25-dihydroxyvitamin D3 and malignant melanoma: the presence of receptors and inhibition of cell growth in culture. Endocrinology 108:1083±1086, 1981
Darwish H, de Luca HF: Vitamin D-regulated gene expression. Crit Rev Eukaryotic Gene Expr 3:89±116, 1993 De Luca HF, Krisinger J, Darwish H: The vitamin D system. Kidney Int 28 (Suppl.):2±8, 1990 Elshourbagy NA, Korman DR, Wu HL, et al: Molecular characterization and regulation of the human endothelin receptors. J Biol Chem 268:3873±3879, 1993 Ermis O, Alpsoy E, Cetin L, et al: Is the ef®cacy of psoralen plus ultraviolet A therapy for vitiligo enhanced by concurrent topical calcipotriol?: a placebo-controlled double-blind study. Br J Dermatol 145:472±475, 2001 Evans SRT, Houghton M, Schumaker L, et al: Vitamin D receptor and growth inhibition by 1,25-dihydroxyvitamin D3 in human malignant melanoma cell lines. J Surg Res 61:127±133, 1996 Frampton RJ, Suva LJ, Eisman JA, et al: Presence of 1,25-dihydroxyvitamin D3 receptors in established human cancer cell lines in culture. Cancer Res 42:1116± 1119, 1982 GlaÈser R, Rowert J, Mrowietz U: Hyperpigmentation due to topical calcipotriol and photochemotherapy in two psoriatic patients. Br J Dermatol 139:148±151, 1998 Haussler MR: Vitamin D receptors. nature and function. Ann Rev Nutr 6:527±562, 1986 Hodgkinson CA, Moore KJ, Nakayama A, et al: Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein. Cell 74:395±404, 1993 Hughes MJ, Lingrel JB, Krakowsky JM, Anderson KP: A helix-loop-helix transcription factor-like gene is located at the mi locus. J Biol Chem 268:20687±20690, 1993 Imokawa G, Yada Y, Miyagishi M: Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes. J Biol Chem 267:24675±24680, 1992 Imokawa G, Miyagishi M, Yada Y: Endothelin-1 as a new melanogen. coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis. J Invest Dermatol 105:32±37, 1995 Imokawa G, Kobayashi T, Miyagishi M, et al: The role of endothelin-1 in epidermal hyperpigmentation and signaling mechanisms of mitogenesis and melanogenesis. Pigment Cell Res 10:218±228, 1997 Ito K, Takeuchi T: The differentiation in vitro of the neural crest cells of the mouse embryo. J Embryol Exp Morphol 84:49±62, 1984 Ito K, Norita T, Sieber-Blum M: In vitro clonal analysis of mouse neural crest development. Dev Biol 157:517±525, 1993 Jimenez M, Tsukamoto K, Hearing VJ: Tyrosinases from two different loci are expressed by normal and by transformed melanocytes. J Biol Chem 266:1147± 1156, 1991 Kawa Y, Ito M, Ono H, et al: Stem cell factor and/or endothelin-3 dependent immortal melanoblast and melanocyte populations derived from mouse neural crest cells. Pigment Cell Res 13 (Suppl. 8):73±80, 2000 Kokelj F, Lavaroni G, Perkan V, et al: Hyperpigmentation due to calcipotriol (MC 903) plus heliotherapy in psoriatic patients. Acta Derm Venereol 75:307±309, 1995 Kovacs SO: Vitiligo. J Am Acad Dermatol 38:647±666, 1998 Lahav R, Ziller C, Dupin E, et al: Endothelin 3 promotes neural crest cell proliferation and mediates a vast increase in melanocyte number in culture. Proc Natl Acad Sci USA 93:3892±3897, 1996 Lahav R, Dupin E, Lecoin L, et al: Endothelin 3 selectively promotes survival and proliferation of neural crest-derived glial and melanocytic precursors in vitro. Proc Natl Acad Sci USA 95:14214±14219, 1998 Lowe KE, Maiyar AC, Norman AW: Vitamin D-mediated gene expression. Crit Rev Eukaryotic Gene Expr 2:65±109, 1992 Mansur CP, Gordon PR, Ray S, et al: Vitamin D, its precursors, and metabolites do not affect melanization of cultured human melanocytes. J Invest Dermatol 91:16±21, 1988 Milde P, Hauser U, Simon T, et al: Expression of 1,25-dihydroxyvitamin D3 receptors and psoriatic skin. J Invest Dermatol 97:230±239, 1991 Murphy M, Reid K, Williams DE, et al: Steel factor is required for maintenance, but not differentiation, of melanocyte precursors in the neural crest. Dev Biol 153:396±401, 1992 Nemere I, Zhou LX, Norman AW: Nontranscriptional effects of steroid hormones. Receptor 3:277±291, 1993 Oikawa A, Nakayasu M: Stimulation of melanogenesis in cultured melanoma cells by calciferols. FEBS Lett 42:32±35, 1974 Ono H, Kawa Y, Asano M, et al: Development of melanocyte progenitors in murine Steel mutant neural crest explants cultured with stem cell factor, endothelin-3, or TPA. Pigment Cell Res 11:291±298, 1998 Ooka S, Kawa Y, Ito M, et al: Establishment and characterization of a mouse neural crest derived cell line (NCCmelan5). Pigment Cell Res 14:268±274, 2001 Parsad D, Saini R, Verma N: Combination of PUVAsol and topical calcipotriol in vitiligo. Dermatology 197:167±170, 1998 Sakurai T, Yanagisawa M, Masaki T: Molecular characterization of endothelin receptors. Trends Pharmacol Sci 13:103±108, 1992 Smith PJW, Teichert-Kuliszewska K, Monge JC, et al: Regulation of endothelin-B receptor mRNA expression in human endothelial cells by cytokines and growth factors. J Cardiovasc Pharmacol 1 (Suppl.):158±160, 1998 Studzinski GP, McLane JA, Uskokovic MR: Signaling pathways for vitamin Dinduced differentiation. implications for therapy of proliferative and neoplastic diseases. Crit Rev Eukaryotic Gene Expr 3:279±312, 1993 Tachibana M: MITF: a stream ¯owing for pigment cells. Pigment Cell Res 13:230± 240, 2000 Tachibana M: Evidence to suggest that expression of MITF induces melanocyte
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differentiation and haploinsuf®ciency of MITF causes Waardenburg syndrome type 2A. Pigment Cell Res 10:25±33, 1997 Tachibana M, Perez-Jurado LA, Nakayama A, et al: Cloning of MITF, the human homolog of the mouse microphthalmia gene and assignment to chromosome 3p14.1-p12.3. Hum Mol Genet 3:553±557, 1994 Tomita Y, Torinuki W, Tagami H: Stimulation of human melanocytes by vitamin D3possibly mediates skin pigmentation after sun exposure. J Invest Dermatol 90:882±884, 1988 Watabe H, Soma Y, Ito M, et al. All-trans retinoic acid induces differentiation and apoptosis of murine melanocyte precursors with induction of the microphthalmia-associated transcription factor. J Invest Dermatol 118:35±42, 2002
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Yada Y, Higuchi K, Imokawa G: Effects of endothelins on signal transduction and proliferation in human melanocytes. J Biol Chem 266:18352±18357, 1991 Yalcin B, Sahin S, Bukulmaz G, et al: Experience with calcipotriol as adjunctive treatment for vitiligo in patients who do not respond to PUVA alone: a preliminary study. J Am Acad Dermatol 44:634±637, 2001 Yasumoto K, Yokoyama K, Shibata K, et al: Microphthalmia-associated transcription factor as a regulator for melanocyte-speci®c transcription of the human tyrosinase gene. Mol Cell Biol 14:8058±8070, 1994 Yasumoto K, Yokoyama K, Takahashi K, et al: Functional analysis of microphthalmia-associated transcription factor in pigment cell-speci®c transcription of the human tyrosinase family genes. J Biol Chem 272:503± 509, 1997
treatment with 1,25-dihydroxyvitamin D3. The induction of endothelin B receptor by 1,25-dihydroxyvitamin D3 was also demonstrated in neural crest cell primary cultures, but not in mature melanocytes. The expression of microphthalmiaassociated transcription factor was induced in NCC-/melb4 cells treated with 1,25-dihydroxyvitamin D3 and endothelin 3, but not by 1,25dihydroxyvitamin D3 alone, suggesting that endothelin 3 may stimulate the expression of the microphthalmia-associated transcription factor gene after binding to the endothelin B receptor induced by 1,25-dihydroxyvitamin D3. These ®ndings suggest a regulatory role for vitamin D3 in melanocyte development and melanogenesis, and may also explain the working mechanism of vitamin D3 in the treatment of vitiligo. Key words: endothelin 3/melanogenesis/microphthalmia-associated transcription factor/ vitamin D receptor/vitamin D3. J Invest Dermatol 118:583±589, 2002
The effects of 1,25-dihydroxyvitamin D3 on the differentiation of immature melanocyte precursors were studied. The NCC-/melb4 cell line is an immature melanocyte cell line established from mouse neural crest cells. 1,25-dihydroxyvitamin D3 inhibited the growth of NCC-/melb4 cells at concentrations higher than 10±8 M. That growth inhibition was accompanied by the induction of tyrosinase and a change in L-3,4-dihydroxyphenylalanine reactivity from negative to positive. Electron microscopy demonstrated that melanosomes were in more advanced stages after 1,25-dihydroxyvitamin D3 treatment. In primary cultures of murine neural crest cells, L-3,4dihydroxyphenylalanine-positive cells were increased after 1,25-dihydroxyvitamin D3 treatment. These ®ndings indicate that 1,25-dihydroxyvitamin D3 stimulates the differentiation of immature melanocyte precursors. Moreover, immunostaining and reverse transcription±polymerase chain reaction analysis revealed that endothelin B receptor expression was induced in NCC-/melb4 cells following
T
(VDR) (Haussler, 1986), which is a member of the superfamily of nuclear steroid/thyroid receptors. After binding 1,25(OH)2D3, the receptors form homodimers and heterodimers with other members of the superfamily and act as transcription factors for a variety of genes (Carlberg, 1993). This is referred to as the classic genomic effect of 1,25(OH)2D3 because it involves gene transcription and new protein synthesis. Vitamin D3 stimulates the differentiation and inhibits the proliferation of many different types of tumor cells, including a variety of melanoma cell lines. Murine B16 melanoma cells treated with vitamin D3 exhibit an increase in tyrosinase activity and melanogenesis (Oikawa and Nakayasu, 1974). A signi®cant growth inhibition was noted in human melanoma cell lines treated with vitamin D3 (Evans et al, 1996). There has been a report showing that vitamin D3 did not affect melanization of cultured human melanocytes (Mansur et al, 1988), but in apparent contrast, Tomita et al (1988) showed that vitamin D3 increased the tyrosinase content of cultured human melanocytes. Topical application of vitamin D3 to the epidermis of mice increases the number of L-3,4dihydroxyphenylalanine (DOPA)-positive melanocytes (AbdelMalek et al, 1988). These ®ndings suggest a possible role for vitamin D3 in modulating melanogenesis, but little is known
he active metabolite of vitamin D, namely 1,25dihydroxyvitamin D3 (1,25(OH)2D3), is known for its regulatory role in calcium homeostasis. The hormone, derived either from 7-dehydrocholesterol by the action of ultraviolet light on the skin or from the diet, facilitates the absorption of calcium from the intestine, its mobilization from bone, and its resorption in the kidney (BurgosTrinidad et al, 1990; De Luca et al, 1990; Studzinski et al, 1993). Apart from its role in calcium uptake, 1,25(OH)2D3 is widely acknowledged to be an important regulator of cell growth and differentiation (Lowe et al, 1992; Darwish and De Luca, 1993; Nemere et al, 1993). The biologic responses elicited by 1,25(OH)2D3 have primarily been attributed to the activation of the nuclear vitamin D receptor Manuscript received May 15, 2002; accepted for publication June 20, 2002 Reprint requests to: Yoshinao Soma, MD, Department of Dermatology, St Marianna University School of Medicine, 2-16-1, Sugao, Miyamae-ku, Kawasaki, 216-8511, Japan. Email: [email protected] Abbreviations: EDN, endothelin; EDNRB, endothelin B receptor; NCC, neural crest cells; SCF, stem cell factor; 1,25(OH)2D3, 1,25dihydroxyvitamin D3; VDR, vitamin D receptor. 0022-202X/02/$15.00
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concerning the effect of 1,25(OH)2D3 on the differentiation of melanocyte precursors or cultured neural crest cells (NCC). We have established an immortal cell population of NCC from WB Sl/+ or +/+ mice, and have designated it as NCC-S4.1 (Kawa et al, 2000). From NCC-S4.1 cells, we obtained a cloned cell line named NCC-melb4 by a single-cell cloning method (Kawa et al, 2000). NCC-melb4 cells are immature melanocyte precursors, as they are positive for tyrosinase-related protein-1 and -2 and KIT, but are negative for tyrosinase, endothelin B receptor (EDNRB), and DOPA reaction (Watabe et al, 2002). They are immortal and stable when maintained in a culture medium supplemented with stem cell factor (SCF). Their growth is not dependent on endothelin 3 (EDN3), probably because of their lack of EDNRB expression. We have also obtained a cloned cell line named NCCmelan5 from C57BL/6 mice (Ooka et al, 2001). NCC-melan5 cells require SCF and EDN3 in the culture medium. They appear to be mature melanocytes, as they are positive for tyrosinase, tyrosinaserelated protein-1 and -2, KIT, EDNRB, and DOPA reactions. Using these cell lines and mouse NCC in a primary culture system, the effects of 1,25(OH)2D3 on the differentiation of the cells and their expression of EDNRB were investigated.
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95°C for 60 s (EDNRB) or at 94°C for 10 s (VDR), annealing at 60°C for 30 s (tyrosinase and MITF), at 60°C for 60 s (EDNRB) or at 58°C for 60 s (VDR), polymerization at 72°C for 60 s (tyrosinase and MITF), at 72°C for 120 s (EDNRB) or at 72°C for 30 s (VDR). The primer sequences used were as follows: Tyrosinase sense primer 5¢-CTATGTCATCCCCACAGGCAC-3¢ Tyrosinase anti-sense primer 5¢-TTACCTGCCAGGAGGAGAAGA-3¢ MITF sense primer 5¢-AGTCACTACCAGGTGCAGAC-3¢ MITF anti-sense primer 5¢-CTTGCTTCAGACTCTGTGGG-3¢ EDNRB sense primer 5¢-CGAGCTGTTGCTTCTTGGAGTAG-3¢ EDNRB anti-sense primer 5¢-AACGGAAGTTGTCATATCCGTGAT-3¢ VDR sense primer 5¢-AGATGACCCTTCTGTGACCC-3¢ VDR anti-sense primer 5¢-AGCTTCTTCAGTCCCACCTG-3¢ b-actin primers were from Promega, Inc. (Madison, WI). PCR products were resolved in 2% agarose gels containing ethidium bromide in parallel with DNA molecular weight markers.
Mice C57BL/6 mice purchased from Japan SLC Co., Ltd. (Hamamatsu, Japan) were mated, and the embryos from those matings were used at 9.5 d postcoitum (E9.5).
Western blot analysis Cells were harvested by scraping, collected by centrifugation, and were then lyzed in cell lysis buffer (1% Triton X-100, 10 mM Tris±HCl, pH 7.4). Cell lysates were separated on 10% sodium dodecyl sulfate polyacrylamide gels. After electrophoresis, proteins on the gel were electroblotted on to nitrocellulose ®lters that were then incubated for 2 h in Tris-buffered saline (100 mM NaCl, 50 mM Tris±HCl, pH 7.2) containing 2.5 mg per ml nonfat powdered milk (Tris-buffered saline milk). The ®lters were then incubated overnight in the presence of the tyrosinase antibody (aPEP7, 1 : 500) and was then reacted with alkaline Anti-phosphatase-labeled anti-rabbit IgG (DAKO) for 90 min. The ®lters were washed in Tris-buffered saline milk and the antigens were detected with 5-bromo-4-chloro-3-indolyl phosphate (Sigma) and nitroblue tetrazodium (Sigma).
Cells and culture conditions NCC-melb4 cells were established using a single cell cloning method from an immortal cell population (NCCS4.1) obtained from NCC derived from WB Sl/+ or +/+ mice (Kawa et al, 2000). NCC-melan5 cells were established from NCC of C57BL/6 mice (Ooka et al, 2001). NCC-melb4 cells were grown in Eagle's modi®ed essential medium supplemented with 5% fetal bovine serum and 50 ng per ml recombinant mouse SCF (kindly supplied by Kirin Brewery, Tokyo, Japan). NCC-melan5 cells were grown in Eagle's modi®ed essential medium containing 15% fetal bovine serum, 50 ng per ml recombinant mouse SCF, and 100 nM EDN3 (Peptide Institute Inc, Osaka, Japan). 1,25(OH)2D3 (BIOMOL Research Laboratories, Inc, Plymouth Meeting, PA) was dissolved in ethanol at a concentration of 10±4 M as a stock solution, and then was added to the culture medium at a ®nal concentration of 10±7 M, unless indicated otherwise.
Statistical analysis
MATERIALS AND METHODS
Proliferation assay Cells were plated at 2000 cells per well in 96-well plates. On the next day various concentrations of 1,25(OH)2D3 were added to the culture medium (day 0). On day 5, the cells were incubated with alamar blue dye (Trek Diagnostic Systems, Inc, Westlake, OH) for 4 h and the ¯uorescence was measured using a Fluoroskan II microplate reader (Labsystems, Helsinki, Finland) Immunohistochemistry The antibody to tyrosinase (aPEP7, which recognizes the carboxy terminus of tyrosinase, Jimenez et al, 1991) were kindly supplied by Dr Hearing (NIH, Bethesda, MD). Cells ®xed with 95% ethanol at room temperature were treated with antibodies against tyrosinase, EDNRB (IBL, Maebashi, Japan) or VDR (Diagnostic System Laboratories, Webster, TX) as described previously (Kawa et al, 2000; Ooka et al, 2001; Watabe et al, 2002). They were then reacted with alkaline phosphatase-labeled anti-rat IgG (Southern Biotechnology Associates, Birmingham, LA) or rabbit IgG (DAKO, Carpinteria, CA) and developed using the New Fuchsin Substrate System (DAKO). DOPA reaction and electron microscopy Cells were ®xed with 2% paraformaldehyde at room temperature and were then treated with 0.1% DOPA (Sigma, St Louis, MO) in 0.1 M phosphate buffer (pH 7.2) for 5 h at 37°C. Nuclei were stained by Nuclear fast red (Merck, Darmstadt, Germany). Electron microscopy and electron microscopic DOPA staining were performed as described elsewhere (Kawa et al, 2000). RNA extraction and reverse transcription±polymerase chain reaction (reverse transcription±PCR) analysis Total RNA was extracted from the cells using an RNA isolation kit (QIAGEN, Hilden, Germany) as described by the manufacturer. cDNA was prepared from 10 mg total RNA using MMLV Reverse Transcriptase (Gibco/BRL, Gaithersburg, MD). PCR reactions consisted of 34 cycles for tyrosinase and microphthalmia-associated transcription factor (MITF), 35 cycles for EDNRB or 40 cycles for VDR. PCR reactions were initiated with a ``cold start''; denaturation at 98°C for 10 s (tyrosinase and MITF), at
NCC culture The NCC culture method was that described by Ito and Takeuchi (1984). Trunk regions posterior to the forelimb buds were dissected from E9.5 embryos of C57BL/6 mice using tungsten needles. The trunks were individually treated with 1% trypsin (Difco, Detroit, MI) in Tyrode's solution for 20 min at 4°C. Trypsinization was terminated by washing with Tyrode's solution containing 10% fetal bovine serum. The tissues were gently pipetted with small pore Pasteur pipettes to separate the neural tubes from other components of the trunks. Neural tubes were explanted individually in 12-well plates with Eagle's modi®ed essential medium containing 15% fetal bovine serum and 50 ng per ml recombinant mouse SCF. The explants were ®xed at 13 d in culture and were then subjected to DOPA reaction or immunohistochemistry. Student's t test was used for statistical analysis.
RESULTS Inhibitory effects of 1,25(OH)2D3 on the proliferation of NCC-melb4 cells We studied whether 1,25(OH)2D3 affected the proliferation of NCC-melb4 cells (Fig 1). Cellular proliferation was inhibited by 1,25(OH)2D3 in a concentration-dependent manner and was markedly inhibited at concentrations higher than 10±8 M. Equivalent amounts of vehicle (ethanol) alone had no effects on the cell growth (data not shown). Differentiation of NCC-melb4 cells induced by 1,25(OH)2D3 As NCC-melb4 cells are immature melanocytes, we considered that the growth inhibition elicited by 1,25(OH)2D3 might be associated with their differentiation. We then examined the effects of 1,25(OH)2D3 on the expression of tyrosinase and DOPA reactivity as markers of differentiation. NCC-melb4 cells were initially negative for tyrosinase and DOPA reactivity (Fig 2a,c). Presumably because NCC-melb4 was a cloned cell line, all cells were negative for both reaction without exception. After 72 h of treatment with 1,25(OH)2D3, some but not all of them became tyrosinase positive and DOPA positive (Fig 2b,d). The induction of tyrosinase by 1,25(OH)2D3 was also demonstrated by western blot analysis (Fig 3a) and by reverse transcription±PCR analysis (Fig 3b). These results suggest that 1,25(OH)2D3 stimulates the differentiation of immature melanocyte precursors. 1,25(OH)2D3 induces EDNRB expression Next we examined whether 1,25(OH)2D3 affects EDNRB expression in NCC-melb4 cells. The cells were originally negative for EDNRB
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Figure 3. Enhanced expression of tyrosinase after 1,25(OH)2D3 treatment. Tyrosinase expression in NCC-melb4 cells before and after the treatment with 10±7 M 1,25(OH)2D3 for 72 h was determined by western blotting (a) and by reverse transcription±PCR analysis (b) as described in Materials and Methods. (b) Lanes 1 and 2: b-actin; lanes 3 and 4: tyrosinase.
Figure 1. 1,25(OH)2D3 inhibits cell proliferation in a concentration-dependent manner. NCC-melb4 cells were plated at 2000 cells per well in 96-well plates and were incubated with various concentrations of 1,25(OH)2D3. After 5 d, cells were incubated with alamar blue dye and the ¯uorescence was measured on a microplate reader. **p < 0.01.
in Fig 4(a,b), 1,25(OH)2D3 induced the expression of EDNRB mRNA in NCC-melb4 cells at 10±7 M and 10±8 M, but not at 10±9 M. We then investigated whether 1,25(OH)2D3 alters EDNRB expression in NCC-melan5 cells, which are EDNRBpositive mature melanocytes (Ooka et al, 2001). As shown in Fig 4(c), 1,25(OH)2D3 had no effect on the expression of EDNRB mRNA in NCC-melan5 cells, although NCC-melb4 cells and NCC-melan5 cells expressed VDR mRNA, as demonstrated by reverse transcription±PCR analysis (Fig 5). Differentiation of NCC-melb4 cells after 1,25(OH)2D3 treatment at the ultrastructural level Electron microscopy and ultrastructural DOPA staining are shown in Fig 6. Before treatment with 1,25(OH)2D3, NCC-melb4 cells contained stage I melanosomes only, none being in more advanced stages, and they were DOPA negative. After 72 h of treatment with 1,25(OH)2D3, stage III±IV melanosomes became apparent, and DOPA staining was positive in the Golgi areas and in melanosomes. The combination of 1,25(OH)2D3 and EDN3 induces MITF expression NCC-melb4 cells were incubated with 1,25(OH)2D3 alone or in the presence of EDN3 for 72 h, and were then tested for MITF expression by reverse transcription± PCR analysis (Fig 7). The combination of 1,25(OH)2D3 and EDN3 induced MITF mRNA expression, whereas 1,25(OH)2D3 alone did not. This suggests a possible role for EDNRB signaling in the induction of MITF, and that EDNRB induced by 1,25(OH)2D3 may effectively transduce signals that stimulate MITF expression.
Figure 2. Effects of 1,25(OH)2D3 on tyrosinase expression, DOPA reaction and EDNRB expression. NCC-melb4 cells before (a,c,e) and after (b,d,f) treatment with 10±7 M 1,25(OH)2D3 for 72 h were immunohistochemically stained for tyrosinase (a,b) or EDNRB (e,f), or were tested for DOPA reaction (c,d). Scale bar = 10 mm.
(Fig 2e), but after 72 h of treatment with 10±7 M 1,25(OH)2D3, some but not all of them became EDNRB positive (Fig 2f). To study the induction of EDNRB by 1,25(OH)2D3 at the molecular level, reverse transcription±PCR analysis was performed. As shown
EDNRB-positive cells and DOPA-positive cells increases in NCC cultures after 1,25(OH)2D3 treatment We investigated the effect of 1,25(OH)2D3 on the NCC primary culture system. Explants from C57BL/6 mice embryos contained VDR-positive cells in NCC around the periphery of the epithelial sheets (data not shown). Before treatment with 1,25(OH)2D3, the explants were negative for EDNRB, but after 1,25(OH)2D3 treatment, EDNRBpositive cells appeared in NCC around the periphery of the epithelial sheets (Fig 8). As shown in Fig 9, an increase in DOPApositive cells was seen following treatment of the NCC cultures with 1,25(OH)2D3. DISCUSSION Various factors, including SCF, EDN3, 12-O-tetradecanoylphorbol-13-acetate, and cholera toxin, have been shown to stimulate the differentiation of melanocyte precursors (Murphy
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Figure 5. Reverse transcription±PCR analysis of VDR mRNA expression in NCC-melb4 cells and in NCC-melan5 cells. Total RNA was reverse-transcribed followed by 40 cycles PCR of cDNA using the primers described in Materials and Methods. Lane 1: b-actin of NCC-melb4 cells; lane 2: b-actin of NCC-melan5 cells; lane 3: VDR of NCC-melb4 cells; lane 4: VDR of NCC-melan5 cells.
Figure 4. EDNRB expression is induced by 1,25(OH)2D3 in NCC-melb4 cells but not in NCC-melan5 cells. (a) NCC-melb4 cells were incubated with or without 10±7 M 1,25(OH)2D3 for 72 h. Total RNA was extracted and analyzed by reverse transcription±PCR. Lanes 1 and 2: b-actin; lanes 3 and 4: EDNRB. (b) NCC-melb4 cells were incubated with various concentrations of 1,25(OH)2D3 for 72 h. Total RNA was extracted and analyzed by reverse transcription±PCR. Lane 1: 10±7 M; lane 2: 10±8 M; lane 3: 10±9 M; lane 4: size marker. (c) NCC-melan5 cells were incubated with or without 10±7 M 1,25(OH)2D3 for 72 h. Total RNA was extracted and analyzed by reverse transcription±PCR. Lanes 1 and 2: b-actin; lanes 3 and 4: EDNRB.
et al, 1992; Ito et al, 1993; Ono et al, 1998), but the effect of 1,25(OH)2D3 on the differentiation of melanocyte precursors and on NCC cultures has not yet been established. VDR expression has been detected in several cancer cell lines, including colon carcinoma, breast carcinoma, and malignant melanoma (Colston et al, 1981; Frampton et al, 1982), and in human melanocytes in vivo (Milde et al, 1991). In this study, we show that VDR mRNA is expressed in two murine NCC lines, i.e., NCC-melb4 and NCCmelan5, and that neural crest explants from C57BL/6 mice embryos contain VDR-positive cells in NCC around the periphery of the epithelial sheets, which suggests a possible role for vitamin D in regulating the proliferation and differentiation of melanocyte precursors. In this study, we show that 1,25(OH)2D3, which is the active metabolite of vitamin D, inhibits the growth of NCC-melb4 cells. That growth inhibition is accompanied by the induction of tyrosinase and a change in DOPA reactivity from negative to positive. Electron microscopy demonstrates the presence of melanosomes at more advanced stages in 1,25(OH)2D3-treated cells, compared with untreated cells. In primary NCC cultures, DOPA-positive cells are increased after 1,25(OH)2D3 treatment. These ®ndings indicate that 1,25(OH)2D3 may stimulate the differentiation of immature melanocyte precursors.
Endothelins (EDN1, EDN2, EDN3) are paracrine signal peptides (21 amino acid residues in length) that bind to at least two subtypes of G-protein-coupled heptahelical receptors (EDNRA and EDNRB). Those two receptors share high sequence homology (Elshourbagy et al, 1993), but can be distinguished by their speci®city; however, EDNRA binds EDN1 and EDN2 with higher af®nity, all three EDN are bound equally well by EDNRB (Sakurai et al, 1992). EDN were shown to be bound by normal human melanocytes that respond with increased proliferation and pigmentation (Yada et al, 1991; Imokawa et al, 1992, 1995, 1997). EDN3 also promotes the survival and proliferation of melanocyte precursors (Lahav et al, 1996, 1998; Ono et al, 1998). The expression of EDNRB is directly regulated by tumor necrosis factor a in human endothelial cells (Smith et al, 1998), but the regulation of EDNRB expression in melanocyte precursors remains unknown. Immunostaining and reverse transcription±PCR analysis shown in this study reveal the induction of EDNRB by 1,25(OH)2D3 in NCC-melb4 cells and also in NCC primary cultures. These ®ndings indicate that 1,25(OH)2D3 acts to promote the expression of EDNRB in immature melanocyte precursors, and they also suggest a possible role for 1,25(OH)2D3 in modulating melanocyte development in vivo. To the best of our knowledge, this is the ®rst study reporting the effect of vitamin D3 on EDNRB expression, and moreover, no other factors have been shown to stimulate EDNRB expression in melanocytes or in immature melanocyte precursors. Recently, we showed that all-trans retinoic acid induces the differentiation of NCC-melb4 cells, but that it did not induce EDNRB (Watabe et al, 2002). We have also studied whether other factors, such as SCF, EDN, 12-O-tetradecanoyl-phorbol-13-acetate, or cholera toxin, induce EDNRB on NCC-melb4 cells and found that none of them did (data not shown). Therefore, the induction of EDNRB on melanocyte precursors may be a speci®c function of 1,25(OH)2D3. As EDNRB is one of the marker molecules for mature melanocytes, vitamin D3 may be an essential factor in melanocyte development. Reverse transcription±PCR analysis showed that 1,25(OH)2D3 had no effect on the expression of EDNRB in NCC-melan5 cells (EDNRB-positive mature melanocytes), suggesting that 1,25(OH)2D3 may stimulate EDNRB expression by immature melanocyte precursors such as NCC-melb4 cells but not in mature melanocytes such as NCCmelan5 cells. MITF is the human homolog of the mouse microphthalmia (mi) gene product, which is a novel transcription factor of the basic± helix±loop±helix (bHLH)±leucine zipper protein family (Hodgkinson et al, 1993; Hughes et al, 1993; Tachibana et al, 1994). MITF is able to direct melanocyte-speci®c transcription of the tyrosinase and tyrosinase-related protein-1 genes (Yasumoto et al, 1994, 1997), and is thought to be involved in regulating the differentiation and maintenance of melanocytes (Tachibana, 1997, 2000). It has been suggested that EDN may regulate MITF function at the protein and promoter levels (Tachibana, 2000). This study demonstrates that 1,25(OH)2D3 combined with EDN3 induces MITF expression in NCC-melb4 cells, whereas 1,25(OH)2D3 alone does not. Thus, EDN3 may induce expression
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Figure 6. Electron microscopy and ultrastructural DOPA staining reveal the development of melanocyte precursors with 1,25(OH)2D3. Electron microscopy (a,c) and electron microscopic DOPA staining (b,d) of NCC-melb4 cells were made before (a,b) and after (c,d) 72 h of treatment with 10±7 M 1,25(OH)2D3. Before the treatment, NCC-melb4 cells contained only stage I melanosomes (small arrow) and were DOPA negative. After the treatment, stage III±IV melanosomes (large arrows) were seen, and both Golgi areas and melanosomes were DOPA-positive. Scale bar = 1 mm.
Figure 7. Induction of MITF mRNA by combined treatment with 1,25(OH)2D3 and EDN3. Reverse transcription±PCR analysis of MITF mRNA from NCC-melb4 cells treated with or without 10±7 M 1,25(OH)2D3 and 100 nM EDN3 for 72 h. Lane 1: no addition; lane 2: 1,25(OH)2D3; lane 3: 1,25(OH)2D3 + EDN3; lane 4: size marker.
of the MITF gene in the presence of 1,25(OH)2D3. It also appears that EDNRB induced by 1,25(OH)2D3 functions as the receptor for EDN3, being capable of signal transduction that effectively stimulates MITF expression. There have been several reports of hyperpigmentation resulting from the combined use of topical calcipotriol (a vitamin D analog) and phototherapy in patients with psoriasis or vitiligo vulgaris (Kokelj et al, 1995; GlaÈser et al, 1998; Parsad et al, 1998). Recent clinical trials demonstrated the ef®cacy of topical calcipotriol combined with psoralen and ultraviolet A in treating vitiligo (Yalcin et al, 2001; Ameen et al, 2001; Ermis et al, 2001); however, the working mechanism of vitamin D in that treatment regimen remains unknown. In the treatment of vitiligo, repigmentation is usually perifollicular (Kovacs, 1998), indicating that melanin is
Figure 8. 1,25(OH)2D3 stimulates the expression of EDNRB in primary NCC cultures. Neural tubes of C57BL/6 mice were explanted and cultured with or without 10±7 M 1,25(OH)2D3. The explants were ®xed at 13 d in culture and were subjected to immunohistochemical staining for EDNRB. Note that numerous EDNRB-positive cells (reddish staining) are seen in 1,25(OH)2D3treated cells (b), but not in nontreated cells (a).
produced by the melanocytes in hair follicles. In our study, 1,25(OH)2D3 induces EDNRB expression in immature melanocytes such as NCC-melb4 cells, but not in mature melanocytes such as NCC-melan5 cells. A melanocyte stem cell-like sub-
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Figure 9. 1,25(OH)2D3 increases DOPA-positive cells in primary NCC cultures. Neural tubes of C57BL/6 mice were explanted and cultured with or without 10±7 M 1,25(OH)2D3. The explants were ®xed at 13 d in culture and were subjected to the DOPA reaction. Note that numerous DOPA-positive cells are seen in 1,25(OH)2D3-treated cells (b), but not in nontreated cells (a).
population is thought to be present in the bulge region of hair follicles (Botchkareva et al, 2001). Taken together, we consider that vitamin D may act to induce immature melanocytes in hair follicles to produce melanin by stimulating their differentiation and their expression of EDNRB. Thus, the effects of 1,25(OH)2D3 presented in this study provide some important clues to understand the roles of vitamin D3 in melanocyte development and melanogenesis. It appears that hyperpigmentation is induced not by a calcipotriol monotherapy, but by a combination of phototherapy plus calcipotriol, as most patients with psoriasis who are treated with calcipotriol do not show any increase in skin pigmentation. Then it is considered that there must be some additional effects of the phototherapy on the melanocyte signaling that are independent of the vitamin D effect. EDN secreted by ultraviolet B-exposed keratinocytes are one of the candidates that cause ultravioletinduced hyperpigmentation (Imokawa et al, 1995, 1997). Further studies using human melanocytes and melanocyte precursors are necessary, as the results with murine cells may not be directly applied to human cells. We would like to sincerely thank Dr Hearing (NIH, Bethesda, MD) for providing us with the antibodies for tyrosinase. We would also like to sincerely thank the Kirin Brewery (Tokyo, Japan) for providing us with SCF. This work was supported by grants from the Scienti®c Research Fund of the Ministry of Education, Science, Sports and Culture, Japan (Grant-in Aid for Scienti®c Research, no. 12670843).
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