ZOOLOGICAL SCIENCE 24: 209–217 (2007)
© 2007 Zoological Society of Japan
Excess Tyrosine Stimulates Eumelanin and Pheomelanin Synthesis in Cultured Slaty Melanocytes from Neonatal Mouse Epidermis Tomohisa Hirobe1,2*, Kazumasa Wakamatsu3 and Shosuke Ito3 1
Radiation Effect Mechanism Research Group, National Institute of Radiological Sciences, Chiba 263-8555, Japan 2 Graduate School of Science and Technology, Chiba University, Chiba 263-8522, Japan 3 Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake 470-1192, Japan
The mouse slaty (Dctslt) mutation is known to reduce the activity of dopachrome tautomerase (DCT). The reduced DCT activity inhibits melanosome maturation and reduces the melanin content in the skin, hair and eyes. It is not known whether eumelanin and pheomelanin synthesis in slaty melanocytes is modulated by melanogenic factors. In this study, to address this point, epidermal melanocytes derived from 0.5-, 3.5- and 7.5-day-old wild-type mice (Dct+/Dct+ at the slaty locus) and from congenic mice mutant (Dctslt/Dctslt at that locus) were cultured in serum-free primary culture with or without additional L-tyrosine (Tyr). The content of melanin was measured by high-performance liquid chromatography in the cultured melanocytes as well as culture supernatants in serum-free primary culture. L-Tyr was found to increase the content of pheomelanin in addition to eumelanin in cultured slaty melanocytes and cuture supernatants at all ages tested. The eumelanin and pheomelanin contents in culture supernatants were greater than in cultured melanocytes. The eumelanin and pheomelanin contents in culture supernatants from 7.5-day-old slaty melanocytes in the presence of L-Tyr were greater than those from wild-type melanocytes. These results suggest that the inhibition of eumelanin synthesis by the slaty mutation can be partly restored by the addition of excess L-Tyr. Eumelanin and pheomelanin may accumulate with difficulty in slaty melanocytes and be easily released from them during skin development. L-Tyr may stimulate this release. Key words: slaty, melanoblast, melanocyte, tyrosine, eumelanin, pheomelanin, skin, development, differentiation, melanogenesis
INTRODUCTION Mouse epidermal melanocytes are known to differentiate around the time of birth (Hirobe, 1984) from undifferentiated precursors, termed melanoblasts, which originate from the neural crest at an embryonic age (Rawles, 1947; Mayer, 1973). They increase in number until 3 or 4 days after birth, and then their numbers decrease (Hirobe, 1984). Differentiated melanocytes produce two types of melanin: brownishblack eumelanin and reddish-yellow pheomelanin (Prota, 1980; Ito, 2003). Although differences exist in molecular size and general properties between eumelanin and pheomelanin, these melanins arise from a common metabolic pathway in which dopaquinone is a key intermediate (Prota, 1980; Hearing and Tsukamoto, 1991; Ito, 2003). The mouse slaty (Dctslt or slt) mutation is a recessive autosomal mutation (chromosome 14, about 5 cM from pie* Corresponding author. Phone: +81-43-206-3253/3133; Fax : +81-43-206-4638; E-mail :
[email protected] doi:10.2108/zsj.24.209
bald [s]) (Silvers, 1979). This mutation occurred in a heterogeneous stock carrying limb-deformity (ldJ) and mahogany (mg) (Silvers, 1979). On a nonagouti background, slaty homozygotes possess a slightly diluted coat and slightly yellowish ears (Green, 1972). In addition to the original slaty mutation, two other mutations, slaty light (DctSlt-lt or Sltlt) and slaty 2J (Dctslt-2J or slt2J), have been identified (Budd and Jackson, 1995). The DctSlt-lt mutation has a more severe effect on coat color and is semidominant, and the mouse mutant for Dctslt-2J is a similar phenotype to that for Dctslt (Budd and Jackson, 1995). The slaty mutation is known to change an arginine to a glutamine in the first copper-binding domain of dopachrome tautomerase (DCT), which converts dopachrome (DC) to 5, 6-dihydroxyindole-2-carboxylic acid (DHICA) in the eumelanin synthesis pathway (Korner and Pawelek, 1980; Jackson et al., 1992; Tsukamoto et al., 1992), and to yield about 10 to 30% activity of wild-type DCT in eye extracts (Jackson et al., 1992). DCT was originally identified as tyrosinase (TYR)-related protein-2 (TRP-2) that maps to the mouse slaty locus (Jackson et al., 1992) and has DCT activity (Tsukamoto et al., 1992). DCT is produced from both wild-type and slaty mutant cDNA, but the protein
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level of DCT in the slaty mutant is greatly reduced (Kroumpouzos et al., 1994). Our previous study (Hirobe et al., 2006) showed that the differentiation (initiation of eumelanin and pheomelanin synthesis) of neonatal mouse melanocytes was greatly inhibited by the slaty mutation, whereas cell proliferation was not influenced. However, it is not known whether the differentiation of slaty melanocytes is modulated by melanogenic factors such as L-tyrosine (Tyr). L-Tyr, the starting material of melanin synthesis, is known to stimulate the differentiation of epidermal melanocytes derived from mutant mice with coat-color dilution (Hirobe et al., 2002). However, it is not known whether L-Tyr stimulates the differentiation of epidermal melanocytes derived from slaty mice. These circumstances prompted us to investigate in detail the effect of LTyr on the differentiation of slaty melanocytes from neonatal mouse epidermis in serum-free primary culture by light microscopy using cytochemical techniques. Chemical analysis of eumelanin and pheomelanin in cultured melanocytes
and culture supernatants from neonatal epidermis of wildtype and slaty mice was also performed. MATERIALS AND METHODS Mice All animals used in this study belonged to Mus musculus strain C57BL/10JHir-Dct+/Dct+ (wild-type, black) and its congenic strain C57BL/10JHir-Dctslt/Dctslt (mutant, slaty). C57BL/6J-Dctslt/Dctslt mouse (kindly supplied by Dr. M. L. Lamoreux, Texas A & M University, College Station, TX, USA) was crossed with C57BL/10JHirDct+/Dct+, and the congenic C57BL/10JHir-Dctslt/Dctslt mouse strain was established by continued backcrossing nine times followed by sib mating. The genic constitution of the line differs only at the slaty locus (Hirobe, 2003; unpublished results). The two strains of mice were given water and a commercial diet, OA-2 (Clea Japan, Tokyo, Japan), ad libitum. They were maintained at 24±1°C with 40–60% relative humidity; 12 hr of fluorescent light was provided daily. This study was approved by the ethics committee of the National Institute of Radiological Sciences in accordance with the guidelines of the National Institute of Health.
Fig. 1. Melanoblasts and melanocytes in pure cultures derived from epidermal cell suspensions of 0.5- (A, B), 3.5- (C, D), and 7.5- (E, F) dayold Dctslt/Dctslt mice cultured in MDMD with (B, D, F) or without (A, C, E) 1 mM L-Tyr. After 14 days in culture. L-Tyr stimulated the pigmentation in Dctslt/Dctslt melanocytes at all ages tested. Phase-contrast microscopy. Bar, 100 μm.
Slaty Melanocytes and Tyrosine
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Fig. 2. Kinetics of the proliferation of epidermal melanoblasts and melanocytes from 0.5- (A, D), 3.5- (B, E), and 7.5- (C, F) day-old Dct+/Dct+ (A, B, C) and Dctslt/Dctslt (D, E, F) mice in MDMD with L-Tyr at concentrations of 0 (○), 0.1 (●), 0.5 (△), 1 (▲), and 2 (□) mM. Pure cultures of melanoblasts and melanocytes were obtained after 14 days. The number of melanoblasts and melanocytes was counted by phase-contrast and bright-field microscopy at 1, 7, and 14 days after initiation of the primary culture. The data are the averages of results from three experiments. Each experiment was performed with different litters of mice. Bars indicate the standard error of the mean (SEM) and are shown only when they were larger than the symbols.
Fig. 3. Kinetics of the differentiation of epidermal melanocytes from 0.5- (A, D), 3.5- (B, E), and 7.5- (C, F) day-old Dct+/Dct+ (A, B, C) and Dctslt/Dctslt (D, E, F) mice in MDMD with L-Tyr at concentrations of 0 ( ○), 0.1 ( ●), 0.5 (△), 1 ( ▲), and 2 ( □) mM. Pure cultures of melanoblasts and melanocytes were obtained after 14 days. The number of melanoblasts and melanocytes was counted and the percentage of melanocytes in the melanoblast-melanocyte population was calculated. Protocols are as detailed for Fig. 2.
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Melanocyte primary culture The source of tissue for the culture of melanocytes was dorsal skin from 0.5-, 3.5- and 7.5-day-old mice. Unless stated otherwise, all reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The method for obtaining epidermal cell suspensions was reported previously (Hirobe, 1992). Disaggregated epidermal cell suspensions were pelleted by centrifugation and suspended in Ham’s F-10 medium (Gibco, Grand Island, NY, USA). The cell pellets after centrifugation were resuspended in melanocyte-proliferation medium (MDMD) consisting of Ham’s F-10 medium supplemented with 10 μg/ml insulin (bovine), 0.5 mg/ml bovine serum albumin (Fraction V), 1 μM ethanolamine, 1 μM phosphoethanolamine, 10 nM sodium selenite, 0.5 mM dibutyryl adenosine 3’:5’cyclic monophosphate, 100 U/ml penicillin G, 100 μg/ml streptomycin sulfate, 50 μg/ml gentamycin sulfate and 0.25 μg/ml amphotericin B. The same lots of these supplements were used in this study. The cells in the epidermal cell suspensions were counted in a hemocytometer chamber and plated onto dishes coated with type I collagen (Becton Dickinson, Bedford, MA, USA) at an initial density of 1×106 cells/35-mm dish (1.04×105 cells/cm2). Cultures were incubated at 37°C in a humidified atmosphere composed of 5% CO2 and 95% air (pH 7.2). Medium was replaced by fresh medium four times a week. After 14 days, almost pure cultures of melanoblasts and melanocytes were obtained. In some cases, additional L-Tyr was added to the medium at various concentrations from 0.1 to 2 mM from the initiation of the primary culture. The standard concentration of L-Tyr in Ham’s F-10 medium is 10 μM. Assays for proliferation and differentiation The number of melanoblasts and melanocytes per dish was determined by phase-contrast and bright-field microscopy, and the calculation was based on the average number of cells from 10 randomly chosen microscopic fields covering an area of 0.581 mm2. Bipolar, tripolar, dendritic, polygonal or epithelioid cells, as seen by phase-contrast, which contained brown or black pigment granules,
as observed by bright-field microscopy, were scored as pigmented melanocytes. In contrast, bipolar, tripolar, dendritic or polygonal cells, as seen by phase-contrast, which contained no pigments and were negative to dopa (no tyrosinase activity), were scored as melanoblasts. These cells were stained by the combined dopa-premelanin reaction (combined dopa-ammoniacal silver nitrate staining; Mishima, 1960; Hirobe, 1984). This preferential staining reveals undifferentiated melanoblasts that contain stage I and II melanosomes without TYR activity in addition to TYR-containing differentiated melanocytes. The ammoniacal silver nitrate reaction specifically reveals unmelanized melanosomes as well as melanized melanosomes in melanocytes, the metallic silver particles being deposited with a high degree of selectivity (Mishima, 1960; Hirobe, 1984). Melanoblasts were also stained by antibodies to TRP-1 and DCT (Hirobe et al., 2006). A “melanoblast” is defined here as an unpigmented cell that possesses no TYR activity. The statistical significance of the differences was determined by Student’s t-test for comparisons of groups of equal size. Dopa and combined dopa-premelanin reactions The methods for the dopa and combined dopa-premelanin reactions for cultured melanoblasts and melanocytes were reported previously (Hirobe, 1992). Assays of eumelanin and pheomelanin Epidermal cell suspensions were cultured in MDMD for 12 days. At this time, cultures consisted of mostly pure melanoblasts and melanocytes. They were then provided with 1 ml of fresh MDMD per 35 mm dish. The cells were incubated for a further 2 days, and the resulting culture supernatants from multiple paired dishes (10–20) were collected with a Pasteur pipette and stored in plastic centrifuge tubes (Becton Dickinson) at –80°C. At the same time, cultured melanocytes were harvested with 0.05% trypsin (Difco, Sparks, MD, USA) and 0.02% ethylenediaminetetraacetate in Ca2+- and Mg2+-free phosphate-buffered saline (pH 7.4) at 37°C
Fig. 4. Dopa (A, B) and dopa-premelanin (C, D) reactions of melanoblasts and melanocytes in pure culture of epidermal cell suspensions from Dctslt/Dctslt mice. After 14 days in culture, cells were fixed and incubated with dopa solution (A, B). After the dopa reaction, cells were incubated with ammoniacal silver nitrate solution (C, D). L-Tyr (1 mM) increased the dopa and silver depositions in Dctslt/Dctslt melanoblasts and melanocytes. Bright-field microscopy. Bar, 100 μm.
Slaty Melanocytes and Tyrosine for 10 min. Cell pellets were collected by centrifugation. Samples of the collected cells and culture supernatants were processed for the chemical analysis of eumelanin to detect the specific degradation product pyrrole-2,3,5-tricarboxylic acid (PTCA) (Ito and Fujita, 1985), and of pheomelanin to detect the specific degradation product 4-amino-3-hydroxyphenylalanine (4-AHP) (Wakamatsu et al., 2002), as reported previously (Hirobe et al., 1998, 2004).
RESULTS Melanocyte culture Within 1 day after initiation of the primary culture of epidermal cell suspensions from 0.5-day-old wild-type mice in MDMD, small bipolar or tripolar melanoblasts were scattered between keratinocyte colonies. After 2–3 days, pigment granules appeared. After 4–5 days, melanocytes increased in number. They were more pigmented than before and extended dendrites into the surrounding keratinocytes. After
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8–9 days, the keratinocyte colonies gradually decreased in number, and by 14 days, cultures contained pure melanocytes with intense pigmentation. When epidermal cell suspensions from 0.5-day-old mutant mice were cultured in MDMD, keratinocytes, melanoblasts and melanocytes had proliferated similarly to those from 0.5-day-old wild-type mice. However, the differentiation of melanocytes was greatly inhibited (melanoblasts ca. 50% and melanocytes 50% at 14 days) (Fig. 1A). When epidermal cell suspensions from 0.5-day-old wildtype mice were cultured in MDMD supplemented with various concentrations of L-Tyr, keratinocytes similarly increased in number compared with control cultures without L-Tyr. However, the proliferation of melanocytes was markedly inhibited in a concentration-dependent manner (Fig. 2A). In contrast, the differentiation of wild-type melanocytes
Fig. 5. Changes in the percentage of cells positive to dopa reaction (A, C) as well as to combined dopa-premelanin reaction (B, D) in Dct+/Dct+ (A, B) and Dctslt/Dctslt (C, D) mice at various ages after birth. Cells were cultured for 14 days in MDMD with or without L-Tyr (1mM). L-Tyr increased both percentages in Dctslt/Dctslt mice. The protocols are as detailed for Fig. 2. * shows statistical difference (P
