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Delayed ERK activation by ceramide reduces melanin synthesis in human melanocytes
Cellular Signaling 14 (2002) 779 – 785 www.elsevier.com/locate/cellsig
Delayed ERK activation by ceramide reduces melanin synthesis in human melanocytes Dong-Seok Kim, Sook-Young Kim, Jin-Ho Chung, Kyu-Han Kim, Hee-Chul Eun, Kyoung-Chan Park* Department of Dermatology and Artificial Organ Laboratory of Clinical Research Institute, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-744, Republic of Korea Received 16 November 2001; accepted 26 January 2002
Abstract Sphingolipid metabolites regulate many aspects of cell growth and differentiation. However, the effects of sphingolipids on the growth and melanogenesis of human melanocytes are not known. In the present study, we investigated the effects of sphingolipid metabolites and the possible signalling pathways involved in human melanocytes. Our data show that C2-ceramide inhibits cell growth in a dose-dependent manner, whereas sphingosine-1-phosphate (SPP) has no effect. Moreover, we observed that the melanin content of the cells was significantly decreased by C2-ceramide. The pigmentation-inhibiting effect of C2-ceramide at 1 – 10 AM was stronger than that of kojic acid, tested at 1 – 100 AM. The tyrosinase activity of cell extracts was reduced by C2-ceramide treatment. However, in the cell-free system, C2-ceramide could not suppress tyrosinase, whereas kojic acid directly inhibited tyrosinase. These results suggest that C2-ceramide decreases the pigmentation of melanocytes indirectly regulating tyrosinase. Furthermore, we found that C2-ceramide decreased the protein expression of microphthalmiaassociated transcription factor (MITF), which is required for tyrosinase expression. To identify the signalling pathway of ceramide, we studied the ability of C2-ceramide to influence extracellular signal-regulated protein kinase (ERK) and Akt/protein kinase B (PKB) activation. C2-ceramide induced a delayed activation of ERK ( > 1 h) and a much later activation of Akt/PKB (>3 h) in human melanocytes. In addition, the specific inhibition of the ERK and the Akt signalling pathways by PD98059 and LY294002, respectively, increased melanin synthesis. Thus, it seems that sustained ERK and Akt activation may lead to the suppression of cell growth and melanogenesis. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Ceramide; ERK; Akt/PKB; MITF; Tyrosinase; Melanogenesis; Proliferation
1. Introduction Ceramide and sphingosine-1-phosphate (SPP) are important sphingolipid metabolites and belong to a novel class of lipid second messengers [1]. Ceramide, a metabolite of sphingomyelin, may participate in the regulation of cell growth and differentiation [2,3]. The antiproliferative effect of ceramide was first identified in human promyelocytic leukemia HL-60 cells [4], and on the other hand, ceramide was found to stimulate proliferation in confluent quiescent Swiss 3T3 fibroblasts [3]. Furthermore, ceramide is important in the monocytic differentiation of HL-60 cells induced Abbreviations: SPP, sphingosine-1-phosphate; ERK, extracellular signal-regulated protein kinase; PKB, protein kinase B; MITF, microphthalmia-associated transcription factor. * Corresponding author. Tel.: +82-2-3668-7474; fax: +82-2-3675-1187. E-mail address: [email protected] (K.-C. Park).
by 1a,25-dihydroxyvitamin D3 [5], and it was found to induce the differentiation of the keratinocyte-related cell line DJM-1 [6]. SPP, a further metabolite of ceramide, is a mitogen for many cell types and can regulate calcium mobilization, cell differentiation, and survival [7]. SPP stimulates quiescent Swiss 3T3 fibroblasts in a protein kinase C-independent manner [8]. However, SPP did not influence cell growth in mouse melanoma B16 or human arterial smooth muscle cells [9]. Sphingomyelin is the most common sphingolipid in the skin and ceramides are products of sphingomyelin hydrolysis by sphingomyelinases [10]. Hence, it is conceivable that sphingolipid breakdown products play important roles in the regulation of epidermal proliferation, differentiation, and melanin synthesis. Although many studies have shown the broad-spectrum biological effects of sphingolipids in various types of cells, the actions of sphingolipids in
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melanocytes have received little attention. Tumour necrosis factor-a activates sphingomyelinases, and leads to ceramide accumulation in cells [1]. Recently, it has been reported that tumour necrosis factor-a inhibits the tyrosinase activity and down-regulates melanogenesis in B16 melanoma cells [11]. Alpha-galactosylceramide, a ceramide derivative, was found to have strong antitumour activity in mice with melanoma B16 cell pulmonary metastasis [12], and we have recently found that ceramide regulates cell growth and melanogenesis in an immortalized mouse melanocyte cell line, Mel-Ab [13]. Despite the increasing possibility that ceramide can suppress melanin synthesis, there has been no attempt to test the actions of ceramide in primary cultured melanocytes. Thus, we undertook to investigate the effects of cellpermeable ceramide and SPP on cell growth and melanogenesis using cultured normal human melanocytes. To examine the possible signalling pathways involving ceramide, we also studied changes of extracellular signal-regulated protein kinase (ERK) and Akt/protein kinase B (PKB) signalling induced by C2-ceramide.
noylphorbol-13-acetate (Sigma), 5 Ag/ml insulin (Sigma), 0.5 Ag/ml transferrin (Sigma), 1 Ag/ml tocopherol (Sigma), 1 ng/ml human recombinant basic fibroblast growth factor (Gibco BRL), and 1% penicillin –streptomycin (10,000 U/ ml and 10,000 Ag/ml, respectively; Gibco BRL). The cells were maintained in a humidified incubator with 5% CO2 at 37jC. Second passage melanocytes were used in the experiments. 2.4. Cell viability assay Cell viability was determined by crystal violet assay [16]. After incubation with the test substances for 24 h, the culture medium was removed and replaced with 0.1% crystal violet in 10% ethanol. Cells were stained for 5 min at room temperature and then rinsed four times. The crystal violet retained by the adherent cells was then extracted with 95% ethanol. Absorbance was determined at 590 nm using an ELISA reader (Molecular Devices, Sunnyvale, CA). 2.5. DNA synthesis
2. Materials and methods 2.1. Materials [methyl-3H]Thymidine was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). LY294002 was purchased from Calbiochem (San Diego, CA) and PD98059 was from Cell Signalling Technology (Beverly, MA). C2ceramide (N-acetyl-D-erythro-sphingosine) and SPP were obtained from Alexis (San Diego, CA). Fatty acid-free bovine serum albumin (BSA), kojic acid, synthetic melanin, L-DOPA, and mushroom tyrosinase were purchased from Sigma (St. Louis, MO). Ceramide and SPP were added to cells as a complex with 0.4% BSA. 2.2. Antibodies Phosphorylated Akt (Ser-473, #9271S), phosphorylated Erk-1/2 (Thr-202/Tyr-204, #9101S), and Erk-1/2 (#9102) were purchased from New England Biolabs (Beverly, MA). Akt (c-20, #sc-1618) and actin (C-2) were from Santa Cruz Biotech (Santa Cruz, CA). Microphthalmia Ab-3 (#MS773-P0) and tyrosinase Ab-1 (#MS-800-P1) were obtained from NeoMarkers (Fremont, CA). 2.3. Cell cultures Human epidermal melanocytes were isolated from adolescent foreskins according to the method of Eisinger and Marko [14]. The cells were maintained in modified MCDB 153 (Sigma) as previously described by Medrano and Nordlund [15], which was supplemented with 5% FBS (Hyclone, Logan, UT), 13 ng/ml bovine pituitary extract (Gibco BRL, Gaithersburg, MD), 10 ng/ml 12-O-tetradeca-
The cells (5 104 cells/well) were grown in 24-well plates. Cultured cells were pulsed with 1 ACi of [methyl-3H] thymidine/well. After 24 h, the medium was removed and cells were washed twice with PBS and precipitated with icecold trichloroacetic acid (5%). The precipitated material was dissolved in 0.3 N NaOH and the incorporated [methyl-3H] thymidine was determined using a liquid scintillation counter (MicroBeta Trilux, Wallac Oy, Turku, Finland). 2.6. Measurement of melanin contents Melanin contents were measured according to the method of Tsuboi et al. [17] with a slight modification. Briefly, cells were treated with sphingolipids or kojic acid for 5 days, and cell pellets containing a known number of cells (usually around 1 106) were dissolved in 1 ml of 1 N NaOH at 100jC for 30 min and centrifuged for 20 min at 16,000 g. The optical densities (OD) of the supernatants were measured at 400 nm using an ELISA reader. Standard curves of synthetic melanin (0 –300 Ag/ml) were prepared in triplicate for each experiment. 2.7. Tyrosinase activity Tyrosinase activity was determined by the method described by Tomita et al. [18] with slight modification. Briefly, melanocytes were plated at a density of 20,000 cells/ well in 96-well plates. After incubating with the test substances, the cells were washed with PBS and lysed with 1% Triton-X/PBS (90 Al/well) and then frozen at 80jC for 30 min. After thawing and mixing, 10 Al of 10 mM L-DOPA was added to each well. Following incubation at 37jC, the absorbance was measured every 10 min for at least 1 h at 475 nm. Data were standardized with mushroom tyrosinase.
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To test the direct effects on tyrosinase activity, we used a cell-free assay system. Eighty microliters of PBS containing various concentrations of test substances were mixed with 10 Al of 10 Ag/ml mushroom tyrosinase and 10 Al of 10 mM L-DOPA. Following incubation at 37jC for 30 min, the absorbance was measured at 475 nm. 2.8. Western blot analysis Cells were collected and lysed in cell lysis buffer (62.5 mM Tris – HCl (pH 6.8), 5% h-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride, 2% SDS, and 10 mM EDTA). Ten micrograms of protein per lane were separated by SDS-PAGE and blotted onto nitrocellulose membrane. The nitrocellulose membranes were saturated with 5% dried milk in Tris-buffered saline containing 0.4% Tween 20. Blots were incubated with primary antibodies at a dilution of 1:500 and then further incubated with horseradish peroxidase-conjugated secondary antibody. Bound antibodies were detected using an enhanced chemiluminescence plus kit (Amersham International, Little Chalfont, UK). 2.9. Statistics Differences between results were assessed for significance using the Student’s t test.
3. Results 3.1. Effect of C2-ceramide on DNA synthesis in melanocytes Melanocytes were cultured for 72 h with C2-ceramide or SPP. As shown in Fig. 1, C2-ceramide dose-dependently inhibited DNA synthesis significantly, whereas SPP had no
Fig. 2. Effects of SPP, C2-ceramide, and kojic acid on melanin synthesis in melanocytes. Cells were cultured with 1 – 10 AM SPP, C2-ceramide, or 1 – 100 AM kojic acid for 5 days, and the melanin content was measured as described in the Materials and Methods. Results are the average of three independent experiments F S.D. * * P < .01, * P < .05 compared to control.
effect. Significant inhibition was found at concentrations >5 AM, and a 38% reduction of [3H]thymidine incorporation was observed when C2-ceramide was used at a concentration of 10 AM. Concentrations of C2-ceramide of less than 1 AM did not influence cell proliferation (data not shown). C2-ceramide was not cytotoxic to human melanocytes by cell viability assay at the concentrations used (data not shown). 3.2. Effect of C2-ceramide on melanin synthesis in melanocytes To assess the effects of sphingolipids on melanogenesis, we measured the melanin contents of human melanocytes cultured in the presence of test substances at concentrations in the range of 1– 10 AM. In melanocytes, a constitutive level of pigment was readily detected (10 – 20 pg/cell). As shown in Fig. 2, the pigmentation of melanocytes was significantly inhibited, in a dose-dependent manner, from a C2-ceramide concentration of 1 AM, but SPP showed no significant effect on melanin synthesis. To compare the inhibitory effects of C2-ceramide, we also used kojic acid at concentrations in the range of 1– 100 AM. As expected, the treatment of cells with kojic acid caused a reduction in pigmentation. Interestingly, the inhibitory effect of C2ceramide on melanogenesis was stronger than that of kojic acid. 3.3. Effect of C2-ceramide on tyrosinase activity in melanocytes
Fig. 1. Effects of SPP and C2-ceramide on the proliferation of melanocytes. Cells were treated with 1 – 10 AM SPP or C2-ceramide for 72 h, as described in the Materials and Methods. The results are shown as [3H]thymidine incorporation versus the control (100%). The values are means F S.D. of triplicate wells. * * P < .01 compared to control.
To investigate the mechanisms possibly responsible for the decreased pigmentation induced by C2-ceramide, the tyrosinase activity was examined. A significant dosedependent decrease in tyrosinase activity was induced by
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that of kojic acid on tyrosinase in a cell-free system, as described in the Materials and Methods. As shown in Fig. 4, kojic acid inhibited tyrosinase activity significantly in the cell-free system. In contrast, C2-ceramide did not suppress mushroom tyrosinase, indicating that C2-ceramide does not inhibit tyrosinase activity directly. 3.5. C2-ceramide inhibits MITF and tyrosinase production in melanocytes
Fig. 3. Effects of C2-ceramide and kojic acid on tyrosinase activity in melanocytes. Cells were cultured with 1 – 10 AM C2-ceramide or 1 – 100 AM kojic acid for 3 days, and tyrosinase activity was measured as described in the Materials and Methods. The values represent the means of four independent experiments F S.D. * * P < .01, * P < .05 compared to control.
C2-ceramide (Fig. 3). As expected, melanin reduction by C2-ceramide was accompanied by a parallel decrease in tyrosinase activity. Moreover, the effect of C2-ceramide (1– 10 AM) on tyrosinase activity was stronger than that of kojic acid at concentrations in the 1 –100 AM range. These results indicate that C2-ceramide regulates tyrosinase, and subsequently inhibits melanin synthesis in melanocytes. 3.4. Effect of C2-ceramide on tyrosinase activity in a cellfree system To determine whether C2-ceramide could inhibit tyrosinase directly, we compared the effect of C2-ceramide with
Fig. 4. Effects of C2-ceramide and kojic acid on tyrosinase activity in a cellfree system. After adding 1 – 10 AM C2-ceramide or 1 – 100 AM kojic acid to 96-well plates, tyrosinase activity was measured directly, as described in the Materials and Methods. The values shown represent the means of four independent experiments F S.D. * * P < .01 compared to control.
Recently, it has been reported that microphthalmia-associated transcription factor (MITF) plays an important role in tyrosinase expression. To test whether C2-ceramide does regulate MITF expression, melanocytes were treated with 10 AM of C2-ceramide for 72 h. MITF and tyrosinase levels were assayed by Western blot (Fig. 5). It was found that C2-ceramide treatment led to a reduced level of MITF protein in human melanocytes, and MITF levels began to decrease within 24 h of C2-ceramide stimulation and became almost undetectable after 72 h. Furthermore, a slight decrease of tyrosinase was found with treatment of C2ceramide. 3.6. Delayed activation of ERK and Akt/PKB by C2ceramide in melanocytes In parallel with examining the effects of C2-ceramide on cell proliferation and melanogenesis, we examined the signalling pathway of C2-ceramide. Because C2-ceramide has an antiproliferative effect on melanocytes, we investigated whether this is related to the regulation of ERK and/ or Akt/PKB. Fig. 6 shows a Western blot assay of melanocytes treated with 10 AM C2-ceramide. Surprisingly, a strong and long lasting activation of ERK1 and ERK2 (ERK1/2) (>60 min) was induced by ceramide addition in melanocytes, whereas Akt/PKB activation was much more delayed (>3 h). These results indicate that C2-ceramide
Fig. 5. Decreased MITF protein by C2-ceramide. Melanocytes were cultured in melanocyte growth media and treated with 10 AM of C2ceramide for the indicated periods of time. Equivalent amounts of lysate protein (10 Ag) were used for Western blotting, and Western blots were analysed for MITF and tyrosinase as described in the Materials and Methods.
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Fig. 6. Delayed activation of ERK1/2 and Akt by C2-ceramide. Melanocytes were cultured in melanocyte growth media and treated with 10 AM of C2-ceramide for the indicated times. Serum-starved cells were used as a negative control (NC). Equivalent amounts of lysate protein (10 Ag) were used for Western blotting. The phosphorylation states of Akt and ERK were analysed using phospho-specific Akt and ERK antibody. Equal protein loading was checked using actin antibody.
Fig. 8. Effects of PD98059 pretreatment on the activation of ERK1/2 by C2ceramide. Cells were cultured in melanocyte growth medium and treated with 10 AM C2-ceramide (C2C) for 6 h. Control cells (—) were treated with the vehicle only for the same time. To study ERK pathway inhibition, 20 AM of PD98059 (PD) was preincubated for 1 h. Western blots were analysed to quantify and to determine the phosphorylation state of ERK1/2.
induces the delayed phosphorylation of ERK and Akt/PKB in cultured normal human melanocytes.
Akt signalling pathway. As shown in Fig. 7, melanin synthesis was clearly induced after treatment with PD98059, but LY294002 promoted melanogenesis only slightly. We next wanted to determine whether PD98059 inhibits ERK activation in human melanocytes, and indeed PD98059 was found to block ERK signalling in C2-ceramide treated cells and in normal cells, indicating that the inhibition of the ERK pathway induces melanin synthesis (Fig. 8).
3.7. Effect of PD98059 and LY294002 on melanin synthesis in melanocytes Since the activation of ERK and Akt seemed to decrease melanin synthesis, we examined whether the ERK and/or the Akt signalling pathways were involved in melanogenesis. Therefore, we investigated melanin synthesis after treating with PD98059, a selective inhibitor of MEK (MAPK/ERK kinase) that is a specific upstream activator of ERK, to study the role of ERK in the ceramide-induced inhibition of melanin production. We also used LY294002, a phosphatidylinositol 3 kinase (PI 3-kinase) inhibitor, which blocks the
Fig. 7. Melanin contents of cultured human melanocytes after treatment with PD98059 and LY294002. Melanocytes were cultured with 20 AM PD98059 (PD) and 20 AM LY294002 (LY) for 5 days. Melanin content was subsequently measured as described in the Materials and Methods, and the total melanin quantity expressed in micrograms of melanin/mg of total protein. Results are the means of three independent experiments F S.D. * * P < .01, * P < .05 compared to control.
4. Discussion Several studies have indicated that both ceramide and SPP are involved in cell proliferation, cell differentiation, and apoptosis [7,19]. These sphingolipid metabolites are known to exhibit pleiotrophic functions, which are cell type-dependent [8,10,20,21]. In the present study, we show that C2ceramide inhibits the proliferation of melanocytes, whereas SPP has no effect. There are different intracellular targets for ceramide depending on cell types. Several potential downstream targets of ceramide have been proposed; these include ceramide-activated protein phosphatase [22], ceramide-activated protein kinase [23], and protein kinase C-~ [24]. The present study was initially undertaken to test the hypothesis that ceramide may regulate cell growth signalling via the ERK or the Akt/PKB pathway in melanocytes, because C2-ceramide is well known to induce cell cycle arrest and apoptosis by inhibiting the ERK and the Akt signalling pathways. The role of the ERK signalling pathway in cellular proliferation has been well established. For example, epidermal growth factor stimulates transient ERK activation, which leads to a mitogenic response [25]. Ceramide is known to suppress growth factor- or sphingosine-induced ERK activation and cell proliferation [26]. However, surprisingly, our study shows that C2-ceramide induces a strong and long-lasting activation of ERK1/2, although C2-ceramide strongly inhibited the proliferation of melanocytes. TPA
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is a well-known inducer of ERK activity and has been shown to stimulate the proliferation of many different cell lines [27]. However, TPA leads to an inhibition of proliferation in other cell types, including MCF-7 cells, although ERK is clearly activated [28]. It has been proposed that the duration of ERK activity is important in determining cellular response and that prolonged activation of the ERK pathway may induce cell growth arrest [29]. Hence, it is likely that C2-ceramide may inhibit the proliferation of human melanocytes via the delayed and sustained activation of ERK. Recently, it has been shown that Akt/PKB plays an important role in cell growth regulation and apoptosis inhibition [30,31]. Akt/PKB is activated after cells are exposed to insulin or other growth factors, and its activation occurs via a pathway that includes PI 3-kinase [32]. Interestingly, Akt/PKB was stimulated at a later stage by C2-ceramide in melanocytes. Many types of neuronal cells are known to depend on the PI 3-kinase/Akt kinase pathway as a crucial growth-promoting and anti-apoptotic signal [33]. Since melanocytes are derived from the neuroectoderm, it is imaginable that the Akt kinase pathway also plays an important role in melanocyte proliferation. Recently, it has been reported that ceramide acts upstream of Akt kinase [33]. However, our results show that cell growth inhibition by C2-ceramide was accompanied by the delayed activation of Akt/PKB. This observation raises the question of whether Akt activation plays a role both in the growth inhibition and in the growth induction, depending on different kinetics. Melanogenesis is a major function of melanocytes. To investigate the effect of sphingolipids on melanin production, we treated melanocytes with C2-ceramide and SPP and observed a dramatic inhibition of melanin content upon increasing the concentration of C2-ceramide in the culture medium, whereas SPP had no effect on melanogenesis. We then compared the effect of ceramide with that of kojic acid, since kojic acid is well known to affect melanin formation in melanocytes and melanoma cells [34]. Our results show that the inhibitory effect of C2-ceramide on melanin was stronger than that of kojic acid. To study the action of C2-ceramide on melanogenesis, we investigated the effect of C2-ceramide on tyrosinase activity. Tyrosinase is known to be the rate-limiting enzyme in the process of melanin synthesis. Ceramide was found to inhibit tyrosinase activity significantly in a dose-dependent manner; this effect of C2-ceramide was stronger than that of kojic acid. Interestingly, C2-ceramide could not suppress tyrosinase activity in the cell-free system, whereas kojic acid inhibits tyrosinase directly. These results suggest that the decreased melanogenesis due to C2-ceramide should be attributed to its inhibitory action upon the signalling pathway regulating tyrosinase activity. Earlier studies have shown that the activation of Akt is responsible for the suppression of melanin production in G361 melanoma cells [35] and that the specific inhibition of the Akt pathway by LY294002 stimulates melanin synthesis in B16 melanoma
cells [36]. We showed here that C2-ceramide induced Akt activation and inhibited melanin synthesis. Therefore, we expected that signal transduction through Akt/PKB could be related to decreased melanin content. In our study, LY294002 treatment significantly increased melanin synthesis in human melanocytes. Thus, our findings are in agreement with previous studies, which found that the Akt/ PKB pathway might be involved in melanogenesis of human melanocytes. MITF plays a critical role in melanogenesis as the major transcriptional regulator of tyrosinase [37]. Our results show that C2-ceramide reduced MITF protein levels, although only a slight decrease in tyrosinase was detected. Recent studies demonstrated that the phosphorylation of MITF at serine 73 is responsible for MITF ubiquitination and degradation [38] and that a specific inhibitor of the ERK pathway, PD98059, increased melanin synthesis [39]. These findings suggest that sustained ERK activation can suppress melanogenesis via increased MITF degradation, which is induced by ERK-dependent MITF phosphorylation [40]. In present study, we have shown that C2-ceramide induced prolonged ERK activation and reduced melanin synthesis. Moreover, PD98059 treatment, which blocked ERK activation, increased melanin synthesis more than LY294002 treatment in human melanocytes. In addition, the results of the present study show that C2-ceramide activated ERK more dramatically than Akt, indicating that the ERK pathway plays a more important role in regulating melanin synthesis. Therefore, we suggest that delayed ERK activation may cause MITF degradation, and that this finally results in reduced melanin synthesis. In summary, we have demonstrated that C2-ceramide inhibits cell proliferation and melanogenesis in human melanocytes, and leads to the activation of ERK and the Akt/PKB pathway. These data suggest that delayed ERK and Akt kinase activation may play an important role in the antiproliferative effect of ceramide. Moreover, our results demonstrate that C2-ceramide decreases the pigmentation of melanocytes by reducing MITF and inhibiting tyrosinase activity, which we suggest may be the result of sustained ERK and Akt activation.
Acknowledgements This study was supported by Grant No. 03-2001-042-0 from the SNUH Research Fund. The authors also wish to acknowledge the financial support of the Pacific Corporation.
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Delayed ERK activation by ceramide reduces melanin synthesis in human melanocytes Dong-Seok Kim, Sook-Young Kim, Jin-Ho Chung, Kyu-Han Kim, Hee-Chul Eun, Kyoung-Chan Park* Department of Dermatology and Artificial Organ Laboratory of Clinical Research Institute, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-744, Republic of Korea Received 16 November 2001; accepted 26 January 2002
Abstract Sphingolipid metabolites regulate many aspects of cell growth and differentiation. However, the effects of sphingolipids on the growth and melanogenesis of human melanocytes are not known. In the present study, we investigated the effects of sphingolipid metabolites and the possible signalling pathways involved in human melanocytes. Our data show that C2-ceramide inhibits cell growth in a dose-dependent manner, whereas sphingosine-1-phosphate (SPP) has no effect. Moreover, we observed that the melanin content of the cells was significantly decreased by C2-ceramide. The pigmentation-inhibiting effect of C2-ceramide at 1 – 10 AM was stronger than that of kojic acid, tested at 1 – 100 AM. The tyrosinase activity of cell extracts was reduced by C2-ceramide treatment. However, in the cell-free system, C2-ceramide could not suppress tyrosinase, whereas kojic acid directly inhibited tyrosinase. These results suggest that C2-ceramide decreases the pigmentation of melanocytes indirectly regulating tyrosinase. Furthermore, we found that C2-ceramide decreased the protein expression of microphthalmiaassociated transcription factor (MITF), which is required for tyrosinase expression. To identify the signalling pathway of ceramide, we studied the ability of C2-ceramide to influence extracellular signal-regulated protein kinase (ERK) and Akt/protein kinase B (PKB) activation. C2-ceramide induced a delayed activation of ERK ( > 1 h) and a much later activation of Akt/PKB (>3 h) in human melanocytes. In addition, the specific inhibition of the ERK and the Akt signalling pathways by PD98059 and LY294002, respectively, increased melanin synthesis. Thus, it seems that sustained ERK and Akt activation may lead to the suppression of cell growth and melanogenesis. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Ceramide; ERK; Akt/PKB; MITF; Tyrosinase; Melanogenesis; Proliferation
1. Introduction Ceramide and sphingosine-1-phosphate (SPP) are important sphingolipid metabolites and belong to a novel class of lipid second messengers [1]. Ceramide, a metabolite of sphingomyelin, may participate in the regulation of cell growth and differentiation [2,3]. The antiproliferative effect of ceramide was first identified in human promyelocytic leukemia HL-60 cells [4], and on the other hand, ceramide was found to stimulate proliferation in confluent quiescent Swiss 3T3 fibroblasts [3]. Furthermore, ceramide is important in the monocytic differentiation of HL-60 cells induced Abbreviations: SPP, sphingosine-1-phosphate; ERK, extracellular signal-regulated protein kinase; PKB, protein kinase B; MITF, microphthalmia-associated transcription factor. * Corresponding author. Tel.: +82-2-3668-7474; fax: +82-2-3675-1187. E-mail address: [email protected] (K.-C. Park).
by 1a,25-dihydroxyvitamin D3 [5], and it was found to induce the differentiation of the keratinocyte-related cell line DJM-1 [6]. SPP, a further metabolite of ceramide, is a mitogen for many cell types and can regulate calcium mobilization, cell differentiation, and survival [7]. SPP stimulates quiescent Swiss 3T3 fibroblasts in a protein kinase C-independent manner [8]. However, SPP did not influence cell growth in mouse melanoma B16 or human arterial smooth muscle cells [9]. Sphingomyelin is the most common sphingolipid in the skin and ceramides are products of sphingomyelin hydrolysis by sphingomyelinases [10]. Hence, it is conceivable that sphingolipid breakdown products play important roles in the regulation of epidermal proliferation, differentiation, and melanin synthesis. Although many studies have shown the broad-spectrum biological effects of sphingolipids in various types of cells, the actions of sphingolipids in
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melanocytes have received little attention. Tumour necrosis factor-a activates sphingomyelinases, and leads to ceramide accumulation in cells [1]. Recently, it has been reported that tumour necrosis factor-a inhibits the tyrosinase activity and down-regulates melanogenesis in B16 melanoma cells [11]. Alpha-galactosylceramide, a ceramide derivative, was found to have strong antitumour activity in mice with melanoma B16 cell pulmonary metastasis [12], and we have recently found that ceramide regulates cell growth and melanogenesis in an immortalized mouse melanocyte cell line, Mel-Ab [13]. Despite the increasing possibility that ceramide can suppress melanin synthesis, there has been no attempt to test the actions of ceramide in primary cultured melanocytes. Thus, we undertook to investigate the effects of cellpermeable ceramide and SPP on cell growth and melanogenesis using cultured normal human melanocytes. To examine the possible signalling pathways involving ceramide, we also studied changes of extracellular signal-regulated protein kinase (ERK) and Akt/protein kinase B (PKB) signalling induced by C2-ceramide.
noylphorbol-13-acetate (Sigma), 5 Ag/ml insulin (Sigma), 0.5 Ag/ml transferrin (Sigma), 1 Ag/ml tocopherol (Sigma), 1 ng/ml human recombinant basic fibroblast growth factor (Gibco BRL), and 1% penicillin –streptomycin (10,000 U/ ml and 10,000 Ag/ml, respectively; Gibco BRL). The cells were maintained in a humidified incubator with 5% CO2 at 37jC. Second passage melanocytes were used in the experiments. 2.4. Cell viability assay Cell viability was determined by crystal violet assay [16]. After incubation with the test substances for 24 h, the culture medium was removed and replaced with 0.1% crystal violet in 10% ethanol. Cells were stained for 5 min at room temperature and then rinsed four times. The crystal violet retained by the adherent cells was then extracted with 95% ethanol. Absorbance was determined at 590 nm using an ELISA reader (Molecular Devices, Sunnyvale, CA). 2.5. DNA synthesis
2. Materials and methods 2.1. Materials [methyl-3H]Thymidine was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). LY294002 was purchased from Calbiochem (San Diego, CA) and PD98059 was from Cell Signalling Technology (Beverly, MA). C2ceramide (N-acetyl-D-erythro-sphingosine) and SPP were obtained from Alexis (San Diego, CA). Fatty acid-free bovine serum albumin (BSA), kojic acid, synthetic melanin, L-DOPA, and mushroom tyrosinase were purchased from Sigma (St. Louis, MO). Ceramide and SPP were added to cells as a complex with 0.4% BSA. 2.2. Antibodies Phosphorylated Akt (Ser-473, #9271S), phosphorylated Erk-1/2 (Thr-202/Tyr-204, #9101S), and Erk-1/2 (#9102) were purchased from New England Biolabs (Beverly, MA). Akt (c-20, #sc-1618) and actin (C-2) were from Santa Cruz Biotech (Santa Cruz, CA). Microphthalmia Ab-3 (#MS773-P0) and tyrosinase Ab-1 (#MS-800-P1) were obtained from NeoMarkers (Fremont, CA). 2.3. Cell cultures Human epidermal melanocytes were isolated from adolescent foreskins according to the method of Eisinger and Marko [14]. The cells were maintained in modified MCDB 153 (Sigma) as previously described by Medrano and Nordlund [15], which was supplemented with 5% FBS (Hyclone, Logan, UT), 13 ng/ml bovine pituitary extract (Gibco BRL, Gaithersburg, MD), 10 ng/ml 12-O-tetradeca-
The cells (5 104 cells/well) were grown in 24-well plates. Cultured cells were pulsed with 1 ACi of [methyl-3H] thymidine/well. After 24 h, the medium was removed and cells were washed twice with PBS and precipitated with icecold trichloroacetic acid (5%). The precipitated material was dissolved in 0.3 N NaOH and the incorporated [methyl-3H] thymidine was determined using a liquid scintillation counter (MicroBeta Trilux, Wallac Oy, Turku, Finland). 2.6. Measurement of melanin contents Melanin contents were measured according to the method of Tsuboi et al. [17] with a slight modification. Briefly, cells were treated with sphingolipids or kojic acid for 5 days, and cell pellets containing a known number of cells (usually around 1 106) were dissolved in 1 ml of 1 N NaOH at 100jC for 30 min and centrifuged for 20 min at 16,000 g. The optical densities (OD) of the supernatants were measured at 400 nm using an ELISA reader. Standard curves of synthetic melanin (0 –300 Ag/ml) were prepared in triplicate for each experiment. 2.7. Tyrosinase activity Tyrosinase activity was determined by the method described by Tomita et al. [18] with slight modification. Briefly, melanocytes were plated at a density of 20,000 cells/ well in 96-well plates. After incubating with the test substances, the cells were washed with PBS and lysed with 1% Triton-X/PBS (90 Al/well) and then frozen at 80jC for 30 min. After thawing and mixing, 10 Al of 10 mM L-DOPA was added to each well. Following incubation at 37jC, the absorbance was measured every 10 min for at least 1 h at 475 nm. Data were standardized with mushroom tyrosinase.
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To test the direct effects on tyrosinase activity, we used a cell-free assay system. Eighty microliters of PBS containing various concentrations of test substances were mixed with 10 Al of 10 Ag/ml mushroom tyrosinase and 10 Al of 10 mM L-DOPA. Following incubation at 37jC for 30 min, the absorbance was measured at 475 nm. 2.8. Western blot analysis Cells were collected and lysed in cell lysis buffer (62.5 mM Tris – HCl (pH 6.8), 5% h-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride, 2% SDS, and 10 mM EDTA). Ten micrograms of protein per lane were separated by SDS-PAGE and blotted onto nitrocellulose membrane. The nitrocellulose membranes were saturated with 5% dried milk in Tris-buffered saline containing 0.4% Tween 20. Blots were incubated with primary antibodies at a dilution of 1:500 and then further incubated with horseradish peroxidase-conjugated secondary antibody. Bound antibodies were detected using an enhanced chemiluminescence plus kit (Amersham International, Little Chalfont, UK). 2.9. Statistics Differences between results were assessed for significance using the Student’s t test.
3. Results 3.1. Effect of C2-ceramide on DNA synthesis in melanocytes Melanocytes were cultured for 72 h with C2-ceramide or SPP. As shown in Fig. 1, C2-ceramide dose-dependently inhibited DNA synthesis significantly, whereas SPP had no
Fig. 2. Effects of SPP, C2-ceramide, and kojic acid on melanin synthesis in melanocytes. Cells were cultured with 1 – 10 AM SPP, C2-ceramide, or 1 – 100 AM kojic acid for 5 days, and the melanin content was measured as described in the Materials and Methods. Results are the average of three independent experiments F S.D. * * P < .01, * P < .05 compared to control.
effect. Significant inhibition was found at concentrations >5 AM, and a 38% reduction of [3H]thymidine incorporation was observed when C2-ceramide was used at a concentration of 10 AM. Concentrations of C2-ceramide of less than 1 AM did not influence cell proliferation (data not shown). C2-ceramide was not cytotoxic to human melanocytes by cell viability assay at the concentrations used (data not shown). 3.2. Effect of C2-ceramide on melanin synthesis in melanocytes To assess the effects of sphingolipids on melanogenesis, we measured the melanin contents of human melanocytes cultured in the presence of test substances at concentrations in the range of 1– 10 AM. In melanocytes, a constitutive level of pigment was readily detected (10 – 20 pg/cell). As shown in Fig. 2, the pigmentation of melanocytes was significantly inhibited, in a dose-dependent manner, from a C2-ceramide concentration of 1 AM, but SPP showed no significant effect on melanin synthesis. To compare the inhibitory effects of C2-ceramide, we also used kojic acid at concentrations in the range of 1– 100 AM. As expected, the treatment of cells with kojic acid caused a reduction in pigmentation. Interestingly, the inhibitory effect of C2ceramide on melanogenesis was stronger than that of kojic acid. 3.3. Effect of C2-ceramide on tyrosinase activity in melanocytes
Fig. 1. Effects of SPP and C2-ceramide on the proliferation of melanocytes. Cells were treated with 1 – 10 AM SPP or C2-ceramide for 72 h, as described in the Materials and Methods. The results are shown as [3H]thymidine incorporation versus the control (100%). The values are means F S.D. of triplicate wells. * * P < .01 compared to control.
To investigate the mechanisms possibly responsible for the decreased pigmentation induced by C2-ceramide, the tyrosinase activity was examined. A significant dosedependent decrease in tyrosinase activity was induced by
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that of kojic acid on tyrosinase in a cell-free system, as described in the Materials and Methods. As shown in Fig. 4, kojic acid inhibited tyrosinase activity significantly in the cell-free system. In contrast, C2-ceramide did not suppress mushroom tyrosinase, indicating that C2-ceramide does not inhibit tyrosinase activity directly. 3.5. C2-ceramide inhibits MITF and tyrosinase production in melanocytes
Fig. 3. Effects of C2-ceramide and kojic acid on tyrosinase activity in melanocytes. Cells were cultured with 1 – 10 AM C2-ceramide or 1 – 100 AM kojic acid for 3 days, and tyrosinase activity was measured as described in the Materials and Methods. The values represent the means of four independent experiments F S.D. * * P < .01, * P < .05 compared to control.
C2-ceramide (Fig. 3). As expected, melanin reduction by C2-ceramide was accompanied by a parallel decrease in tyrosinase activity. Moreover, the effect of C2-ceramide (1– 10 AM) on tyrosinase activity was stronger than that of kojic acid at concentrations in the 1 –100 AM range. These results indicate that C2-ceramide regulates tyrosinase, and subsequently inhibits melanin synthesis in melanocytes. 3.4. Effect of C2-ceramide on tyrosinase activity in a cellfree system To determine whether C2-ceramide could inhibit tyrosinase directly, we compared the effect of C2-ceramide with
Fig. 4. Effects of C2-ceramide and kojic acid on tyrosinase activity in a cellfree system. After adding 1 – 10 AM C2-ceramide or 1 – 100 AM kojic acid to 96-well plates, tyrosinase activity was measured directly, as described in the Materials and Methods. The values shown represent the means of four independent experiments F S.D. * * P < .01 compared to control.
Recently, it has been reported that microphthalmia-associated transcription factor (MITF) plays an important role in tyrosinase expression. To test whether C2-ceramide does regulate MITF expression, melanocytes were treated with 10 AM of C2-ceramide for 72 h. MITF and tyrosinase levels were assayed by Western blot (Fig. 5). It was found that C2-ceramide treatment led to a reduced level of MITF protein in human melanocytes, and MITF levels began to decrease within 24 h of C2-ceramide stimulation and became almost undetectable after 72 h. Furthermore, a slight decrease of tyrosinase was found with treatment of C2ceramide. 3.6. Delayed activation of ERK and Akt/PKB by C2ceramide in melanocytes In parallel with examining the effects of C2-ceramide on cell proliferation and melanogenesis, we examined the signalling pathway of C2-ceramide. Because C2-ceramide has an antiproliferative effect on melanocytes, we investigated whether this is related to the regulation of ERK and/ or Akt/PKB. Fig. 6 shows a Western blot assay of melanocytes treated with 10 AM C2-ceramide. Surprisingly, a strong and long lasting activation of ERK1 and ERK2 (ERK1/2) (>60 min) was induced by ceramide addition in melanocytes, whereas Akt/PKB activation was much more delayed (>3 h). These results indicate that C2-ceramide
Fig. 5. Decreased MITF protein by C2-ceramide. Melanocytes were cultured in melanocyte growth media and treated with 10 AM of C2ceramide for the indicated periods of time. Equivalent amounts of lysate protein (10 Ag) were used for Western blotting, and Western blots were analysed for MITF and tyrosinase as described in the Materials and Methods.
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Fig. 6. Delayed activation of ERK1/2 and Akt by C2-ceramide. Melanocytes were cultured in melanocyte growth media and treated with 10 AM of C2-ceramide for the indicated times. Serum-starved cells were used as a negative control (NC). Equivalent amounts of lysate protein (10 Ag) were used for Western blotting. The phosphorylation states of Akt and ERK were analysed using phospho-specific Akt and ERK antibody. Equal protein loading was checked using actin antibody.
Fig. 8. Effects of PD98059 pretreatment on the activation of ERK1/2 by C2ceramide. Cells were cultured in melanocyte growth medium and treated with 10 AM C2-ceramide (C2C) for 6 h. Control cells (—) were treated with the vehicle only for the same time. To study ERK pathway inhibition, 20 AM of PD98059 (PD) was preincubated for 1 h. Western blots were analysed to quantify and to determine the phosphorylation state of ERK1/2.
induces the delayed phosphorylation of ERK and Akt/PKB in cultured normal human melanocytes.
Akt signalling pathway. As shown in Fig. 7, melanin synthesis was clearly induced after treatment with PD98059, but LY294002 promoted melanogenesis only slightly. We next wanted to determine whether PD98059 inhibits ERK activation in human melanocytes, and indeed PD98059 was found to block ERK signalling in C2-ceramide treated cells and in normal cells, indicating that the inhibition of the ERK pathway induces melanin synthesis (Fig. 8).
3.7. Effect of PD98059 and LY294002 on melanin synthesis in melanocytes Since the activation of ERK and Akt seemed to decrease melanin synthesis, we examined whether the ERK and/or the Akt signalling pathways were involved in melanogenesis. Therefore, we investigated melanin synthesis after treating with PD98059, a selective inhibitor of MEK (MAPK/ERK kinase) that is a specific upstream activator of ERK, to study the role of ERK in the ceramide-induced inhibition of melanin production. We also used LY294002, a phosphatidylinositol 3 kinase (PI 3-kinase) inhibitor, which blocks the
Fig. 7. Melanin contents of cultured human melanocytes after treatment with PD98059 and LY294002. Melanocytes were cultured with 20 AM PD98059 (PD) and 20 AM LY294002 (LY) for 5 days. Melanin content was subsequently measured as described in the Materials and Methods, and the total melanin quantity expressed in micrograms of melanin/mg of total protein. Results are the means of three independent experiments F S.D. * * P < .01, * P < .05 compared to control.
4. Discussion Several studies have indicated that both ceramide and SPP are involved in cell proliferation, cell differentiation, and apoptosis [7,19]. These sphingolipid metabolites are known to exhibit pleiotrophic functions, which are cell type-dependent [8,10,20,21]. In the present study, we show that C2ceramide inhibits the proliferation of melanocytes, whereas SPP has no effect. There are different intracellular targets for ceramide depending on cell types. Several potential downstream targets of ceramide have been proposed; these include ceramide-activated protein phosphatase [22], ceramide-activated protein kinase [23], and protein kinase C-~ [24]. The present study was initially undertaken to test the hypothesis that ceramide may regulate cell growth signalling via the ERK or the Akt/PKB pathway in melanocytes, because C2-ceramide is well known to induce cell cycle arrest and apoptosis by inhibiting the ERK and the Akt signalling pathways. The role of the ERK signalling pathway in cellular proliferation has been well established. For example, epidermal growth factor stimulates transient ERK activation, which leads to a mitogenic response [25]. Ceramide is known to suppress growth factor- or sphingosine-induced ERK activation and cell proliferation [26]. However, surprisingly, our study shows that C2-ceramide induces a strong and long-lasting activation of ERK1/2, although C2-ceramide strongly inhibited the proliferation of melanocytes. TPA
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is a well-known inducer of ERK activity and has been shown to stimulate the proliferation of many different cell lines [27]. However, TPA leads to an inhibition of proliferation in other cell types, including MCF-7 cells, although ERK is clearly activated [28]. It has been proposed that the duration of ERK activity is important in determining cellular response and that prolonged activation of the ERK pathway may induce cell growth arrest [29]. Hence, it is likely that C2-ceramide may inhibit the proliferation of human melanocytes via the delayed and sustained activation of ERK. Recently, it has been shown that Akt/PKB plays an important role in cell growth regulation and apoptosis inhibition [30,31]. Akt/PKB is activated after cells are exposed to insulin or other growth factors, and its activation occurs via a pathway that includes PI 3-kinase [32]. Interestingly, Akt/PKB was stimulated at a later stage by C2-ceramide in melanocytes. Many types of neuronal cells are known to depend on the PI 3-kinase/Akt kinase pathway as a crucial growth-promoting and anti-apoptotic signal [33]. Since melanocytes are derived from the neuroectoderm, it is imaginable that the Akt kinase pathway also plays an important role in melanocyte proliferation. Recently, it has been reported that ceramide acts upstream of Akt kinase [33]. However, our results show that cell growth inhibition by C2-ceramide was accompanied by the delayed activation of Akt/PKB. This observation raises the question of whether Akt activation plays a role both in the growth inhibition and in the growth induction, depending on different kinetics. Melanogenesis is a major function of melanocytes. To investigate the effect of sphingolipids on melanin production, we treated melanocytes with C2-ceramide and SPP and observed a dramatic inhibition of melanin content upon increasing the concentration of C2-ceramide in the culture medium, whereas SPP had no effect on melanogenesis. We then compared the effect of ceramide with that of kojic acid, since kojic acid is well known to affect melanin formation in melanocytes and melanoma cells [34]. Our results show that the inhibitory effect of C2-ceramide on melanin was stronger than that of kojic acid. To study the action of C2-ceramide on melanogenesis, we investigated the effect of C2-ceramide on tyrosinase activity. Tyrosinase is known to be the rate-limiting enzyme in the process of melanin synthesis. Ceramide was found to inhibit tyrosinase activity significantly in a dose-dependent manner; this effect of C2-ceramide was stronger than that of kojic acid. Interestingly, C2-ceramide could not suppress tyrosinase activity in the cell-free system, whereas kojic acid inhibits tyrosinase directly. These results suggest that the decreased melanogenesis due to C2-ceramide should be attributed to its inhibitory action upon the signalling pathway regulating tyrosinase activity. Earlier studies have shown that the activation of Akt is responsible for the suppression of melanin production in G361 melanoma cells [35] and that the specific inhibition of the Akt pathway by LY294002 stimulates melanin synthesis in B16 melanoma
cells [36]. We showed here that C2-ceramide induced Akt activation and inhibited melanin synthesis. Therefore, we expected that signal transduction through Akt/PKB could be related to decreased melanin content. In our study, LY294002 treatment significantly increased melanin synthesis in human melanocytes. Thus, our findings are in agreement with previous studies, which found that the Akt/ PKB pathway might be involved in melanogenesis of human melanocytes. MITF plays a critical role in melanogenesis as the major transcriptional regulator of tyrosinase [37]. Our results show that C2-ceramide reduced MITF protein levels, although only a slight decrease in tyrosinase was detected. Recent studies demonstrated that the phosphorylation of MITF at serine 73 is responsible for MITF ubiquitination and degradation [38] and that a specific inhibitor of the ERK pathway, PD98059, increased melanin synthesis [39]. These findings suggest that sustained ERK activation can suppress melanogenesis via increased MITF degradation, which is induced by ERK-dependent MITF phosphorylation [40]. In present study, we have shown that C2-ceramide induced prolonged ERK activation and reduced melanin synthesis. Moreover, PD98059 treatment, which blocked ERK activation, increased melanin synthesis more than LY294002 treatment in human melanocytes. In addition, the results of the present study show that C2-ceramide activated ERK more dramatically than Akt, indicating that the ERK pathway plays a more important role in regulating melanin synthesis. Therefore, we suggest that delayed ERK activation may cause MITF degradation, and that this finally results in reduced melanin synthesis. In summary, we have demonstrated that C2-ceramide inhibits cell proliferation and melanogenesis in human melanocytes, and leads to the activation of ERK and the Akt/PKB pathway. These data suggest that delayed ERK and Akt kinase activation may play an important role in the antiproliferative effect of ceramide. Moreover, our results demonstrate that C2-ceramide decreases the pigmentation of melanocytes by reducing MITF and inhibiting tyrosinase activity, which we suggest may be the result of sustained ERK and Akt activation.
Acknowledgements This study was supported by Grant No. 03-2001-042-0 from the SNUH Research Fund. The authors also wish to acknowledge the financial support of the Pacific Corporation.
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