Enhancement of pheomelanogenesis by L-dopa in the mouse melanocyte cell line, TM10, in vitro

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1 Enhancement of pheomelanogenesis by L-dopa in the mouse melanocyte cell line, TM10, in vitro CHIKARA SATO 1, SHOUSUKE ITO 2 and TAKUJI TAKEUCHI 1 " 'Biological Institute, Tohoku University, Aoba-yama Sendai 980, Japan and z School of Hygiene, Fujita-Cakuen Health University, Toyoake, Aichi , Japan Author for correspondence Summary Cells of TM10, an established cell line, are melanocytes that contain equal amounts of eumelanin (black pigment) and pheomelanin (yellow pigment). The content of pheomelanin drastically increased when the cells were cultured in growth medium containing 0-2 mm-l-dopa (Ldihydroxyphenylalanine), which is the common precursor for both eumelanogenesis and pheomelanogenesis. After this treatment, the amount of pheomelanin was 3-7-fold greater than that of control in TM10, whereas the amount of eumelanin changed very little. In contrast, 5-S-cysteinyldopa, which is the specific precursor for pheomelanogenesis downstream of L-dopa, did not cause preferential increase in pheomelanogenesis. Ultrastructural observations also confirmed these results; in 0-2 mm-l-dopa, an increase in the number of pheomelanosomes was observed in the cytoplasm of TM10 cells. Our results also suggest that the L-dopa treatment results in a decrease in tyrosinase activity per melanosome. Key words: melanocyte, pheomelanin, L-dopa, mouse, tyrosinase. Introduction Mammalian melanocytes are capable of producing two types of melanin, black pigment (eumelanin) and yellow pigment (pheomelanin). In the house mouse, the types of melanin produced in melanocytes are known to be determined by local tissue environment, which is influenced by the agouti (a) locus (Silvers & Russel, 1955; Galbraith et al. 1979; Mayer & Fishbane, 1972). In that process, products of the a locus are considered to be produced in the hair follicle that surrounds melanocytes and to determine the type of melanin through intercellular interactions (Silvers & Russel, 1955; Tamate & Takeuchi, 1984). Yet, the mechanism involved in the induction of the shift in the melanogenesis is still unknown. The B16 mouse melanoma cell line, which has been widely used in the study of melanogenesis, does not seem to be suitable for studying the shift between eumelanogenesis and pheomelanogenesis, for the cells produce eumelanin only (Sato et al. 1985; Ito & Fujita, 1984). It was also shown that the cells possessed no Journal of Cell Science 87, (1987) Printed in Great Britain The Company of Biologists Limited 1987 morphologically normal chromosomes (Ishiguro & Takeuchi, 1981). In an attempt to develop a suitable experimental system for studying the regulation of pheomelanogenesis, we established a melanocyte cell line with normal properties by culturing mouse melanocytes from C57BL/6J mice with PMA (phorbol-12-myristate-13-acetate) and cholera toxin. Among several cell lines established, one of them, TM10, was found to produce both eumelanin and pheomelanin in equal amounts as reported (Sato et al. 1985). In this paper, we describe the fact that the ratio of pheomelanin to eumelanin can be drastically increased by addition of L-dopa as well as L-dopa phosphates to the culture medium, and discuss its relationship to the shift in melanogenesis under normal circumstances in vivo. Materials and methods Culture methods The cell line used in this study was TM10, which was derived from C57BL/6J newborn mice (Satoef al. 1985). Cells were plated in tissue culture dishes (Falcon 3002) at a density of 507

2 1X10 4 cells ml ' in the growth medium (MEM (Eagle's minimum essential medium) supplemented with 10% CS (calf serum, Flow laboratory)). The medium was changed 3 days later. The cells were then cultured in growth medium containing L-dopa, 5-S-cysteinyl-dopa (5-S-C-dopa) or L- dopa phosphates; 72 h later, cells were harvested with 0-05 % trypsin/0-02% EDTA in CMF-PBS (Ca 2+ /Mg 2+ -free phosphate-buffered saline) and washed three times by centrifugation in CMF-PBS. L-dopa phosphates were synthesized by the method reported by Pawelek & Murray (1986). Hitachi 557 two-wavelength/double beam Spectrophotometer. The rate was measured during the first few minutes of the reaction while it was linear. Correction for L-dopa autoxidation was made. Specific activity is defined as formation of dopachrome (absorbance at 475 nm) per min per mg protein in the reaction mixture. Protein was estimated by Miller's (1959) modification of the Lowry method, using a protein standard solution of bovine albumin fraction "V (Wako) in0-lm-naoh. Quantitative analysis of eumelanin and pheomelanin The amounts of eumelanin and pheomelanin were determined according to Ito & Fujita (1984). Permanganate oxidation of eumelanin yields pyrrole-2,3,5-tricarboxylic acid (PTCA), while hydriodic acid hydrolysis of pheomelanin yields aminohydroxyphenylalanine (AHP). PTCA and AHP were analysed by high-performance liquid chromatography. The cellular contents of eumelanin and pheomelanin were obtained by correcting the values of PTCA and AHP using the factors estimated from the yields (Ito & Fujita, 1984). Electron microscopy For electron microscopy, cells were harvested with 005 % trypsin/0-02% EDTA in CMF-PBS. Cell pellets were fixed with 3-5% glutaraldehyde in 0-1 M-phosphate buffer (ph7-4) and postfixed with 1% osmium tetroxide in the same phosphate buffer. After fixation, they were dehydrated through a graded series of ethanol and embedded in Epon 812. The ultrathin sections were stained with uranyl acetate and lead citrate, and examined in a Hitachi HS-9 electron microscope. In order to analyse the ratios of the types of the melanosomes, the numbers of eumelanosomes, pheomelanosomes and mosaic-type melanosomes were recorded for each 100 micrographs of nucleated cells for the control cells and L- dopa-treated cells. The micrographs were enlarged and the types of the melanosomes were categorized according to the ultrastructure in the melanosomes. In order to estimate the two-dimensional density of the melanosomes in the cytoplasm, we recorded the number of the melanosomes for 200 micrographs of nucleated cells for each experiment. It was divided by the area of the cytoplasm. Micrographs were enlarged times, and cytoplasmic areas of the photoprints were cut out. The area of the cytoplasm was determined using an automatic area metre (Hayashi-Denkou AAM-5) after counting the number of the melanosomes. Ultrathin sections were also selected, so as to avoid recording micrographs of the same cell. Tyrosinase activity Tyrosinase activity was assayed as dopa oxidase activity with a slight modification of the method reported by Wrathal et al. (1973). The cell pellet was dissolved in 2ml of 0-5 % sodium desoxycholate in distilled water and allowed to stand at 0 C for 30 min. Tyrosinase activity was analysed spectrophotometrically by following the oxidation of L-dopa to dopachrome at 475 nm. The reaction mixture consisted of 40 ml of 0-04% L-dopa in 0-1 M-phosphate buffer, ph6-8, and 0-4 ml of the extract. Assays were performed at 37 C C with a Results Effects of L-dopa, 5-S-cysteinyl-dopa and L-dopa phosphates on the types of melanogenesis in TMJ0 cells In the pathway of melanin synthesis, L-dopa is the precursor common to both eumelanogenesis and pheomelanogenesis. 5-S-cysteinyl-dopa is the precursor that is located far downstream from L-dopa and specific to pheomelanogenesis. We examined the effects of L-dopa and 5-S-cysteinyl-dopa on melanin production in TM10 cells, which are capable of producing both eumelanin and pheomelanin in MEM supplemented with 10 % CS, in the proportion of 10 to 8 (Sato et al. 1985). After culturing the TM10 cells for 3 days, L- dopa or 5-S-cysteinyl-dopa was added to the growth medium of the cells. Following culture for 72 h, the contents of eumelanin and pheomelanin in the cells were measured. A significant increase in the content of pheomelanin was observed when the concentration of L-dopa was increased. In the cells cultured in 0-2 mm- L-dopa, the amount of pheomelanin was 3-7-fold higher than that of the control, whereas the amount of eumelanin was not affected (Fig. 1A,B). On the other hand, treatment with 5-S-cysteinyl-dopa did not result in such a specific increase in pheomelanogenesis as did L-dopa. In order to exclude the possibility that the products of auto-oxidation of L-dopa in the culture medium are responsible for the enhanced pheomelanogenesis, the effect of L-dopa phosphates on TM10 cells was examined. When the cells were cultured in growth medium containing L-dopa phosphates, a significant increase in pheomelanogenesis was observed (Fig. 2). L-dopa phosphates are not oxidized by serum proteins and generate L-dopa within the cells by removal of the phosphate (Pawelek & Murray, 1986). Therefore, our result seems to indicate that the L-dopa-induced enhancement of pheomelanogenesis is not due to autooxidation in the medium. Electron-microscopic observation In order to examine whether the enhancement of pheomelanogenesis is due to an increase in the number of pheomelanosomes or to an increase in the amount of 508 C. Sato et al.

3 pheomelanin in a melanosome, electron-microscopic observation was performed. TMIO cells were cultured in growth medium containing 0-2mM-L-dopa or in the control growth medium. In the control, both eumelanosomes containing striated longitudinal matrices and pheomelanosomes containing vesiculo-globular structures were observed in the same ratio (Fig. 3). In the cells cultured in 0-2mM-L-dopa, pheomelanosomes were observed more frequently than eumelanosomes (Fig. 4). In both experiments, we observed mosaictype melanosomes: longitudinal matrices of the eumelanosome type and vesiculo-globular bodies of the pheomelanosome type were seen in a single melanosome. The numbers of pheomelanosomes, eumelanosomes and mosaic-type melanosomes were recorded for 100 micrographs of nucleated cells, for control cells and L-dopa-treated cells, respectively. The photomicrographs were enlarged times, and the types of Concentration of L-dopa or 5-5-C-dopa (log IHM) 0-2 Fig. 1. Effect of L-dopa or 5-5-cysteinyl-dopa on the content of pheomelanin and eumelanin in TM10. After 3 days culture in growth medium, cells were cultured in medium containing L-dopa or 5-S-cysteinyl-dopa for 72 h, and were harvested to analyse melanin content. The contents of pheomelanin (A) and eumelanin (B) were obtained by multiplying the contents of AHP and PTCA by factors of 5 and 50, respectively (Ito & Fujita, 1984). ( ) TMlO-treated with L-dopa; (O) TM10 treated with 5-5-cysteinyl-dopa; (A) 3T3 cells treated with L-dopa. A. Content of pheomelanin in the cells. ( ) Each value is the mean ± s.e. of four experiments; (O) each value is the mean ± S.E. of three experiments; (A) each value is the mean of two experiments: B. Content of eumelanin in the cells. Each value represents the mean ± S.E. of four experiments. Pheomelanogenesis in cultured melanocytes 509

4 melanosomes were categorized according to the ultrastructure in the melanosomes. The ratios of three types of melanosomes in each experiment are shown in Table 1. The ratio of pheomelanosomes was 36-8% in 10 the control, whereas it increased to 71-0% in the Ldopa treated cells. On the contrary, the ratio of eumelanosomes decreased from 51-4% to 12-1%. That of mosaic-type melanosomes increased only from 11-8% to 16-9%. In the present experiment, on the other hand, an increase in the number of melanosomes in a cell was also noticed. To estimate two-dimensional densities of the melanosomes, the number of melanosomes in the cytoplasm and the area of the cytoplasm were measured for 200 micrographs of nucleated cells. The average density in the cytoplasm was 0-34 melanosomes/xm~z in the control, whereas, in L-dopa_-*»* Concentration of L-dopa phosphates (logmin) 0-2 Fig. 2. Effect of L-dopa phosphates on the content of pheomelanin and eumelanin in TM10. Culture methods were the same as for Fig. 1. ( ) Content of pheomelanin in the cells. Each value is the mean ± S.E. of three experiments. ( ) Content of eumelanin in the cells. Each value is the mean ± S.E. of four experiments. 9m -t Fig. 4. Electron micrograph of TM10 cells (0-2mM-Ldopa-treated). Numerous melanosomes are seen; most melanosomes are pheomelanosomes. p, pheomelanosome; mo, mosaic-type melanosome; m, mitochondrion; n, nucleus; C, Golgi apparatus. X Table 1. Effect of L-dopa on the constitution of three types of melanosomes in TMI0 cells Fig. 3. Electron micrograph of TM10 cells (control). Eumelanosomes and pheomelanosomes are seen on the same level, e, eumelanosome; m, mitochondrion; n, nucleus; G, Golgi apparatus. X C. Sato et al. Classification of melanosomes Control L-dopa Pheomelanosome Eumelanosome Mosaic-type melanosome 36-8 ± ± ± ± ±2-6 After 3 days culture in growth medium, cells were cultured in medium containing 0-2mM-L-dopa or in control medium, for 72 h. Electron-microscopic observations were performed so as not to record micrographs from the same cell, as described in the text. Each value is the ratio of a type of melanosome to all the melanosomea in the section (mean ± S.E. of 100 cells).

5 treated cells, it increased to 0-91 melanosomes fim 2 (Table 2). These results seem to suggest that the enhancement of pheomelanogenesis is due to the increase in the number of pheomelanosomes. Effects of L-dopa on tyrosinase activity Tyrosinase is known to be the key enzyme in melanogenesis. It catalyses the conversion of tyrosine to L- dopa and that of L-dopa to dopaquinone. Tyrosinase activity was measured using cells treated with various concentrations of L-dopa. It was demonstrated that the activity per cell was not affected by concentrations of L-dopa up to 0-2 mm as shown in Fig. 5. In the cells treated with 0-2mM-L-dopa, two-dimensional density in the cytoplasm was 2-7-fold higher than that in the control (Table 2). Therefore, this seems to suggest that average tyrosinase activity per melanosome was decreased in these circumstances. Discussion It has been reported that pheomelanin was accumulated, following culture in L-dopa-containing medium, in the hair bulbs of explants from 2-day-old wild-type mice with the genotype A/A (Takeuchi, 1971). However, the experimental system was too complex to Concentration of L-dopa (logmm) Fig. 5. Influence of L-dopa on the tyrosinase activity in TM10 cells. The experimental conditions were exactly as described in the text. Each value represents the mean ± S.E. of triplicate experiments. 0-2 determine its precise mechanism. In order to develop a suitable experimental system, we established cultured melanocyte cell lines (Sato et al. 1985). In the present study, L-dopa is shown to induce pheomelanogenesis in one of the cultured melanocyte cell lines, TM10. In contrast, 5-5-cysteinyl-dopa, a direct precursor of pheomelanin, did not enhance pheomelanogenesis. These results seem to indicate that the enhancement of pheomelanogenesis by L-dopa was not caused only by the increase in the concentration of the substrates. Electron-microscopic observations revealed that the enhancement involves an increase in the number of pheomelanosomes in the cell. On the other hand, in O2mM-L-dopa-treated cells, the average tyrosinase activity per melanosome was found to be decreased. This result suggests that the level of tyrosinase activity within a melanosome has some relationship with the type of melanin produced in the melanosome. Although the role of tyrosinase in the change in the type of melanogenesis in vivo is unclear, it has been suggested that low tyrosinase activity is associated with pheomelanogenesis in some mutant mice. The tyrosinase activity of the dorsal skin of a A y /a mouse and a e/e mouse was also shown to be lower than that of the wild type (a/a, E/E) (Lamoreux, personal communication). It seems to be important to pay attention to the level of tyrosinase activity within a melanosome. Although we do not know the precise function of each isozyme of tyrosinase, a change in the isozyme pattern might be involved in this phenomenon. Wongeia/. (1974) reported that a-msh (a'-melanocyte-stimulating hormone) caused an increase in the tyrosinase activity in Cloudman S91 mouse melanoma cells. It was also shown that excessive addition of a- MSH to the melanocytes also caused a change from pheomelanogenesis to eumelanogenesis in vivo (Geshwind et al. 1972) and in vitro (Tamate & Takeuchi, 1981, 1984). It seems possible that enhanced tyrosinase activity within a melanosome caused eumelanogenesis in those experiments. The results presented here and the experimental results that have been accumulated (unpublished) lead to a hypothesis on the type of melanogenesis in vivo. It Table 2. Effect of L-dopa on the two-dimensional density of the melanosomes in the cytoplasm oftmlo cells Control L-dopa Ratio (L-dopa/control) Density of melanosomes (melanosomes ^m~ 2 ) 0-34 ± ± After 3 days culture in growth medium, cells were cultured in medium containing 0'2mM-L-dopa or in control medium, for 72 h. Electron-microscopic observations were performed so as not to record micrographs from the same cell, and areas of the cytoplasm were measured with the auto area metre as described in the text. Each value is the two-dimensional density of the melanosomes in the cytoplasm per section (mean ± S.E. of 200 cells). Significance of difference: P< Pheomelanogenesis in cultured melanocytes 511

6 is also likely that low tyrosinase activity per melanosome is associated with pheomelanogenesis only when the cysteinyl compounds available in the melanosome are sufficient for pheomelanogenesis. TMIO cells seem to provide enough cysteinyl compounds for their melanosomes. In the present study, L-dopa did not affect eumelanogenesis in TMIO cells, whereas eumelanogenesis was decreased in the shift from eumelanogenesis to pheomelanogenesis in vivo in melanocytes with the A allele at the a locus. This difference may be due to the slow translocation of the melanosomes in cultured melanocytes. Melanosomes are accumulated in the cytoplasm of cultured melanocytes, whereas melanosomes are effectively transferred into keratinocytes in the hair follicles. Newly formed melanosomes in cultured melanocytes are added to the melanosomes that already exist in the cytoplasm. There is a possibility that auto-oxidation products of L-dopa are responsible for enhanced pheomelanogenesis. This possibility was excluded in our study by using L-dopa phosphates that had been shown to be stable in the culture medium and to generate L-dopa only within the cells (Pawelek & Murray, 1986). Further investigations to elucidate the mechanism of the shift between the two forms of melanogenesis are awaited, using the unique cell line, TMIO. References GALBRAITH, D. B., WOLFF, G. L. & BREWER, N. L. (1979). Tissue microenvironment and the genetic control of hair pigment patterns in mice. Devi Genet. 1, GESHWIND, I. I., HUSEBY, R. A. & NISHIOKA, R. (1972). The effect of melanocyte stimulating hormone on coat color in the mouse. Recent Prog. Harm. Res. 28, ISHIGURO, S. & TAKEUCHI, T. (1981). Activation and suppression of melanogenesis in mouse melanoma cells by hybridization with chick embryonic cells. Dev. Growth Differ. 23, ITO, S. & FUJITA, K. (1984). Microanalysis of eumelanin and pheomelanin in hair and mouse melanomas by chemical degradation and liquid chromatography. Analyt. Biochem. 144, KORNER, A. & PAWELEK, J. (1982). Mammalian tyrosinase catalyzes three reactions in the biosynthesis of melanin. Science 217, MAYER, T. C. & FISHBANE, J. L. (1972). Mesoderm-ectoderm interaction in the production of the agouti pigmentation pattern in mice. Genetics 71, MILLER, G. L. (1959). Protein determination for large number of samples. Analyt. Chem. 31, 964. PAWELEK, J. M. & MURRAY, M. (1986). Increase in melanin formation and promotion of cytotoxity in cultured melanoma cells caused by phosphorylated isomers of L-dopa. Cancer Res. 46, SATO, C, ITO, S. & TAKEUCHI, T. (1985). Establishment of a mouse melanocyte clone which synthesises both eumelanin and pheomelanin. Cell Struct. Fund. 10, SILVERS, W. K. & RUSSEL, E. S. (1955). An experimental approach to action of genes at the agouti locus of the mouse. J. exp. Zool. 130, TAKEUCHI, T. (1971). Regulating function of agouti gene in the mouse. In Biology of Normal and Abnormal Melanocytes (ed. T. Kawamura, T. B. Fitzpatrick & M. Seiji), pp Tokyo: University of Tokyo Press. TAMATE, H. B. & TAKEUCHI, T. (1981). Induction of the shift in melanin synthesis in lethal yellow (A y /a) mice in vitro. Devi Genet. 2, TAMATE, H. B. & TAKEUCHI, T. (1984). Action of the e locus of mice in the response of pheomelanic hair follicles to ar-melanocyte-stimulating hormone in vitro. Science 224, WONG, G., PAWELEK, J., SANSONE, M. & MOROWTTZ, J. (1974). Response of mouse melanoma cells to melanocyte stimulating hormone. Nature, Lond. 248, WRATHAL, J. R., OLIVER, C, SILAGI, S. & ESSNER, E. (1973). Suppression of pigmentation in mouse melanoma cells by 5-bromodeoxyuridine. J. Cell Biol. 57, (Received 30 September Accepted, in revised form, 27 January 1987) 512 C. Sato et al.