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Chaetocin inhibits IBMX-induced melanogenesis in B16F10 mouse melanoma cells through activation of ERK
Chemico-Biological Interactions 245 (2016) 66e71
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Chaetocin inhibits IBMX-induced melanogenesis in B16F10 mouse melanoma cells through activation of ERK Jung-Soo Bae a, b, c, 1, Mira Han a, b, c, 1, Cheng Yao a, b, c, Jin Ho Chung a, b, c, d, * a
Department of Dermatology, Seoul National University College of Medicine, Seoul, Republic of Korea Laboratory of Cutaneous Aging Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea c Institute of Human-Environment Interface Biology, Seoul National University, Seoul, Republic of Korea d Institute on Aging, Seoul National University, Seoul, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2015 Received in revised form 22 December 2015 Accepted 29 December 2015 Available online 31 December 2015
Chaetocin is a natural product isolated from Chaetomium species that has anti-bacterial and antimyeloma activities. In this study, we investigated the inhibitory effect of chaetocin on melanogenesis and the underlying mechanisms in B16F10 mouse melanoma cells. In the present study, chaetocin significantly inhibited IBMX-induced melanin production and tyrosinase activity without any cytotoxicity. Furthermore, chaetocin down-regulated both the protein and mRNA levels of tyrosinase, which is a specific enzyme that catalyzes the conversion of tyrosine to melanin. We also observed that the protein level of MITF was significantly reduced by chaetocin treatment. In addition, we found that the antimelanogenic effect of chaetocin was suppressed by treatment with the specific ERK inhibitor (PD98059). Accordingly, chaetocin inhibited melanogenesis via suppressing the protein level of MITF followed by activation of the ERK signaling pathway. These data suggest that chaetocin may be a potential anti-melanogenic agent for use in skin-whitening cosmetics and a topical agent for treatment of hyperpigmentation disorders. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Chaetocin Melanin Tyrosinase Microphthalmia-associated transcription factor Extracellular signal-regulated kinases ERK
1. Introduction Melanin is the major pigment synthesized by epidermal melanocytes in the skin and it determines the skin color due to its own color: eumelanin (black) and pheomelanin (yellow). In addition, melanin has an important role in the physiological defense of skin protection from ultraviolet (UV) radiation. However, repetitive UV irradiation induces increased accumulation of melanin and leads to skin hyperpigmentation [1]. Hyperpigmented skin has an unaesthetic appearance such as chloasma and freckles [2,3]. The biosynthesis of melanin is induced by several stimuli such
Abbreviations: a-MSH, a-melanocyte-stimulating hormone; PBS, phosphatebuffered saline; DMEM, Dulbecco's modified Eagle's media; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IBMX, 3-isobutyl-1methylxanthine; MITF, microphthalmia-associated transcription factor; UV, ultraviolet; L-DOPA, L-3,4-dihydroxyphenylalanine; TRP, tyrosinase-related protein; ERK, extracellular signal-regulated kinases. * Corresponding author. Department of Dermatology, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Republic of Korea. E-mail address: [email protected] (J.H. Chung). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.cbi.2015.12.021 0009-2797/© 2015 Elsevier Ireland Ltd. All rights reserved.
as UV radiation, inflammation, hormones, or other skin injuries [4e6]. For instance, inflammation is closely related with melanogenesis, and a large number of inflammatory cytokines have been reported as melanogens. Histamine, an ubiquitous inflammatory mediator, is a representative of melanogen induced by inflammation [7]. In addition, several chemicals elevating intracellular cAMP (forskolin and IBMX), and substrates of melanogenesis such as Ltyrosine and L-dihydroxyphenylalanine (L-DOPA) can also activate melanogenesis [8,9]. Moreover, UV radiation is considered as the primary factor of melanogenesis in normal physiological conditions. UV radiation stimulates cutaneous hypothalamic-pituitaryadrenal (HPA) axis to regulate local stress responses, and produces POMC peptides such as MSH, b-endorphin and ACTH. These peptides increase melanin pigmentation by interacting with melanocortin receptor on melanocytes [10e13]. Endocannabinoids are also generated in keratinocytes in response to UV radiation, and that increases melanin synthesis by activating endocannabinoid signaling pathway [14]. In general, these melanogenic stimuli lead to an increase in microphthalmia-associated transcription factor (MITF) expression. MITF is the master regulator of melanin synthesis which induces the transcription of melanogenic specific enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1),
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and dopachrome tautomerase (DCT) [15]. Therefore, downregulation of these molecules involved in the process of melanin synthesis is regarded as a way of skin whitening. There are various skin whitening agents, such as kojic acid, arbutin, and linoleic acid. Kojic acid and arbutin are widely used in Northeast Asia as cosmetic ingredients due to their anti-tyrosinase activity. Arbutin is a glycosylated hydroquinone extracted from the bearberry plant, and it has generally been used as a treatment for hyperpigmentation disorders in the past [16]. In addition, linoleic acid suppresses tyrosinase activity by accelerating ubiquitindependent degradation of tyrosinase protein [17]. However, some of these agents have been reported to cause skin irritation. It has been reported that kojic acid causes skin irritation and side effects such as cytotoxicity, dermatitis, and skin cancer [8]. Arbutin has been also banned due to its side effects involving exogenous ochronosis and perdurable depigmentation [18,19]. For these reasons, some countries have placed restrictions on the use of these chemicals as cosmetic ingredients. Recently, numerous reports have focused on the development of a safe skin-whitening agent from natural sources due to side effects of the established agents. 6-Shogaol, one of the ingredients present in ginger, decreases melanogenesis by activation of the ERK pathway [20]. Extracts of Artocarpus communis also alleviate aMSH-stimulated melanin accumulation in B16F10 melanoma cells [21]. In addition, Caffeoylserotonin, one of serotonin derivatives from a wide range of plant species inhibits melanogenesis [22]. Chaetocin is one of the natural products isolated from Chaetomium species fungi with antimicrobial and cytostatic activities. However, its pharmacological action on the skin has not been fully understood. In this study, we investigated the anti-melanogenic effect of chaetocin on IBMX-induced melanogenesis in B16F10 mouse melanoma cells as well as explored the underlying molecular mechanisms involved in this process. 2. Materials and methods 2.1. Chemicals and reagents Chaetocin, 3-isobutyl-1-methylxanthine (IBMX), L-DOPA, and 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). The inhibitor PD98059 was obtained from Calbiochem (San Diego, CA, USA). Specific antibodies for phospho-ERK1/2, total ERK1/2, and phospho-AKT were purchased from Cell Signaling (Beverly, MA, USA). Antibodies recognizing tyrosinase (C-19) and actin (I-19) were obtained from Santa Cruz (Santa Cruz, CA, USA). MITF antibody (MS-771-P0) was purchased from NeoMarkers (Fremont, CA, USA). 2.2. Cell culture B16F10 mouse melanoma cell line obtained from the Korean Cell Line Bank (Seoul, Korea) was cultured in Dulbecco's modified Eagle's media (DMEM; Gibco-BRL, Carlsbad, CA, USA) without phenol red, supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) and penicillin/streptomycin (400 U/mL, 50 g/L) at 37 C under the humidified condition with 5% CO2. To maintain cell characteristics, cells were split every 2 days. 2.3. Cell viability assay B16F10 cell viability was determined using MTT-based assay. B16F10 cells (2.5 103 cells/well) were seeded into 96-well plates and incubated with chaetocin (10, 20, 30, or 40 nM) for 24 h and 48 h, respectively. After incubation, culture plates were treated
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with MTT (dissolved in PBS to 0.5 g/L) for 4 h. The supernatant was removed and the formazan crystals were dissolved in DMSO. The amount of formazan was quantified at 570 nm using ELISA reader (Thermo Fisher Scientific Inc., USA). 2.4. Measurement of melanin content Measurement of intracellular melanin content was quantified by using the previously described method with slight modification [20]. B16F10 cells were stimulated by IBMX and incubated with chaetocin or the inhibitor PD98059 at the same time for 48 h. The cell pellets were harvested and then dissolved in 1 N NaOH containing 10% DMSO at 80 C for 1 h. The melanin content was analyzed at 475 nm using ELISA reader. For accurate calculation of melanin content, each level of melanin was normalized to the protein content. 2.5. Tyrosinase activity Tyrosinase activity was determined using the method described previously [24]. B16F10 cells were first treated with IBMX and further treated with serial concentrations of chaetocin at the same time. After 48 h incubation, the cells were washed with cold PBS and lysed in phosphate saline (pH 6.8) containing Triton-X100 (1.0%) and protease inhibitor cocktail (Roche Diagnostics). Cell lysates were centrifuged and then the protein content of the supernatant was normalized using the Bradford protein assay. Then, 90 mL of cell extract was placed in a 98-well plate treated with freshly prepared 10 mL of L-DOPA (final concentration of 1 mmol/L) in 25 mM phosphate buffer (pH 6.8) and incubated at 37 C for 20 min. Absorbance was measured at 475 nm using an ELISA reader. 2.6. Western blot analysis B16F10 cells (2.5 105 cells) were seeded into 60 mm dishes and incubated with chaetocin. Cells were washed with cold PBS and homogenized in 50 mM TriseHCl (pH 7.4) buffer containing 1% NP40, 0.25% deoxycholic acid, 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma Aldrich). Protein contents of total cell lysates were determined using BCA protein assay (Sigma Aldrich). Each sample was adjusted to same concentration and 20 mg of protein per lane was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a PVDF membrane. The membrane was blocked with 5% fat-free milk in TBST (Tris-buffered saline containing 0.4% Tween 20) followed by incubation with appropriate primary antibodies. After 24 h incubation with primary antibodies, blots were further incubated for 1 h with horseradish peroxidase-conjugated secondary antibody. Immune complexes were visualized using an enhanced chemiluminescence detection system (GE Healthcare). 2.7. Reverse transcription and real-time quantitative PCR Total RNA was isolated from B16F10 cells using RNAiso Plus (Takara Bio Inc., Shiga, Japan), as previously described [25]. Isolated RNAs were reverse-transcribed into cDNAs using the First Strand cDNA Synthesis Kit (MBI Fermentas, Vilnius, Lithuania). The synthesized cDNAs were subjected to real-time quantitative PCR and quantitation of PCR products was performed by 7500 Real time PCR System (Applied Biosystems, Foster City, CA) with the SYBR Premix Ex Taq II kit (Takara Bio). The PCR reactions were performed with the primers for MITF, tyrosinase, TRP-1, and endogenous reference 36B4. Data were analyzed using the 2DDCT method and data are expressed as the fold number of gene expression.
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2.8. Statistical analyses All data were analyzed using Microsoft Excel 2007 software. Results are expressed as means ± SEM, and statistical analyses were performed using a Student's t-test. P-values of less than 0.05 were considered statistically significant.
decreased the expression levels of tyrosinase protein (Fig. 2D) and mRNA (Fig. 2E). In addition, we observed that the level of TRP-1 mRNA was decreased by chaetocin treatment (Fig. 2F). These results indicate that chaetocin has an anti-melanogenic effect on B16F10 mouse melanoma cells. 3.3. Chaetocin inhibits MITF protein expression
3. Results 3.1. Cytotoxicity of chaetocin in B16F10 mouse melanoma cells To investigate whether chaetocin has cytotoxic effects, we treated B16F10 mouse melanoma cells with various doses of chaetocin in the absence or presence of IBMX. The viability of B16F10 cells was measured using the MTT-based assay. The cell viability was measured at 48 h after treatment with 10, 20, 30, or 40 nM chaetocin. We observed that chaetocin had no significant effect on cell viability either in the absence (Fig. 1A) or presence (Fig. 1B) of IBMX. The results showed that chaetocin has little cytotoxic effect in B16F10 cells at the concentrations used in the present study. 3.2. Chaetocin inhibits IBMX-induced melanin synthesis in B16F10 mouse melanoma cells We next investigated the effect of chaetocin on IBMX-induced melanogenesis. We demonstrated that chaetocin inhibited IBMXinduced melanin synthesis in a dose-dependent manner (Fig. 2A, B). Tyrosinase is a well-known catalyzing enzyme which mediates melanin production. Therefore, we investigated the effects of chaetocin on tyrosinase enzymatic activity and tyrosinase expression after chaetocin treatment. Chaetocin significantly decreased the IBMX-induced tyrosinase activity in a dose-dependent manner (Fig. 2C). We also observed that treatment with chaetocin
To understand the transcriptional regulation of tyrosinase and TRP-1 mRNA expression, we investigated the effect of chaetocin on the expression of MITF, which is a major transcription factor for tyrosinase and TRP-1. After treatment with chaetocin, the protein level of MITF was down-regulated in a dose-dependent manner (Fig. 3A). Chaetocin treatment also decreased the IBMX-induced MITF protein expression in a time-dependent manner (Fig. 3B). These results indicate that chaetocin has inhibitory activity on MITF protein expression. 3.4. Suppression of the ERK pathway relieved chaetocin-induced inhibition of melanogenesis To understand how chaetocin regulates MITF expression, we investigated the time-dependent effects of chaetocin on MITF expression and phosphorylation of ERK and AKT. We confirmed that chaetocin decreased the IBMX-induced MITF protein expression (Fig. 4A). On the other hand, chaetocin increased the phosphorylation of ERK, but not of AKT. To investigate whether increased phosphorylation of ERK is involved in inhibition of MITF production, pretreatment with a selective inhibitor of the ERK pathway, PD98059, was performed before chaetocin treatment. We found that treatment with PD98059 relieved chaetocin-induced inhibition of MITF expression. Phosphorylation of ERK induced by chaetocin was also inhibited by PD98059 (Fig. 4B). To further investigate the effect of chaetocin-induced ERK phosphorylation on
Fig. 1. Effects of chaetocin on cell viability in B16F10 mouse melanoma cells. Cell viability was analyzed by MTT assay. B16F10 cells were treated for 48 h with (A) chaetocin at a concentration of 10, 20, 30, or 40 nM and (B) chaetocin plus IBMX (100 mM). (C) The chemical structure of chaetocin. Each bar represents the mean ± SEM of three independent experiments.
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Fig. 2. Effect of chaetocin on IBMX-induced melanin production and tyrosinase activity. B16F10 mouse melanoma cells were co-cultured with chaetocin (10, 20, or 30 nM) and IBMX (100 mM). After incubation for 48 h, the melanin content was analyzed. (A) Photograph of cell culture dishes. (B) Melanin content results are presented as the number of folds. (C) Tyrosinase activity was evaluated by L-DOPA oxidation assay. Tyrosinase protein expression was determined by Western blotting at 24 h after treatment (D). mRNA levels of tyrosinase (E) and TRP-1 (F) were determined by RT-qPCR at 12 h after treatment. All RT-qPCR experiments were performed five times. Each bar represents the mean ± SEM. Asterisks in all graphs denote a value that is statistically different (*, P < 0.05; **, P < 0.01) between the IBMX only and IBMX þ chaetocin treated groups.
Fig. 3. Chaetocin inhibits MITF protein expression without affecting its mRNA level. B16F10 cells were treated with 100 mM IBMX or/and chaetocin (10, 20, or 30 nM) (A) MITF protein expression was measured at 4 h after various doses of chaetocin treatment. (B) MITF protein expression was also checked at different time points after co-treatment with chaetocin (30 nM) and IBMX (100 mM). Protein expression was determined by Western blotting.
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Fig. 4. Suppression of the ERK pathway relieved chaetocin-induced inhibition of melanogenesis. (A) Cells were incubated with IBMX (100 mM) plus chaetocin (30 nM) for the indicated times mentioned above. Changes in MITF, p-AKT, and p-ERK protein expressions were visualized by Western blotting. (B) B16F10 cells were treated with IBMX alone or IBMX plus 30 nM chaetocin in the absence or presence of PD98059 (20 mM). Changes in MITF and phosphorylated ERK expressions were checked at 4 h after treatment. Melanin contents were measured at 48 h after treatment. All results are indicated as the mean ± SEM of four different experiments. Asterisks in the graph denote a value that is statistically different (* and #, P < 0.05).
melanin production, melanin contents were evaluated in the presence of PD98059 in chaetocin-treated cells. The level of melanin inhibited by chaetocin was increased by PD98059 treatment (Fig. 4C). Thus, these results indicated that activation of the ERK pathway is involved in chaetocin-induced reduction of melanogenesis. 4. Discussion Chaetocin is a natural product isolated from Chaetomium species fungi, and it is known as a specific inhibitor of the lysine-specific histone methyltransferase SUV39H1 (suppressor of variegation 3e9 homolog 1) in Drosophila melanogaster [18,19]. Other reports have shown that chaetocin reduces the methylation of H3K9 and H3K27 [26,27]. Chaetocin has potent anti-myeloma activity which is attributable to induction of reactive oxygen species (ROS) imposed by inhibition of thioredoxin reductase [28,29]. However, in spite of many published reports, the effect of chaetocin on melanogenesis has not been studied well. In the present study, we investigated the effect of chaetocin on melanin synthesis and the underlying molecular mechanisms involved in this process in B16F10 mouse melanoma cells. Tyrosinase, a copper-containing enzyme, is an important enzyme for controlling the production of melanin [30]. Hence, molecules controlling tyrosinase expression and activity have been considered as treatment of hyperpigmentation [31]. In this study,
we showed the anti-melanogenic effect of chaetocin in a dosedependent manner without any cytotoxicity in B16F10 mouse melanoma cells. Furthermore, we found that tyrosinase expression and activity were decreased by chaetocin treatment in B16F10 mouse melanoma cells. MITF is a transcription factor that is closely related to the development of melanocytes and osteoclasts. As a major transcription factor in melanogenesis, MITF can control the expression of several genes which act as critical enzymes in the conversion of tyrosine to melanin. In this study, we demonstrated that chaetocin dramatically decreased MITF protein expression. The MAPK/ERK pathway plays an important role in proliferation and differentiation of various types of cells [32,33]. Especially in melanocytes, the ERK pathway can control melanogenesis by regulating MITF expression. Several reports have noted that phosphorylation of ERK attenuated a-MSH and IBMX-induced MITF protein expression at the posttranslational level [27]. Previous reports have demonstrated that the activated ERK induces the phosphorylation of MITF at serine-73 and leads to MITF ubiquitin-dependent degradation [15,34]. The PI3K/Akt signaling pathway also has an important role in melanin synthesis [35]. Activation of PI3K/Akt pathway reduces melanogenesis by regulating tyrosinase transcription in melanocyte. Therefore, we studied the effect of chaetocin on the Akt activation, but observed no significant changes in Akt phosphorylation. On the other hand, chaetocin effectively activated the ERK signaling pathway, and lead to down-regulation of MITF expression.
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Moreover, down-regulated MITF protein levels by chaetocin were restored by inhibiting ERK activation by PD98059. In addition, the inhibitory effect of chaetocin against melanin synthesis was alleviated by inhibiting the ERK pathway. In summary, we identified that chaetocin inhibited melanogenesis in IBMX-treated B16F10 cells without any cytotoxicity. We found that chaetocin induced ERK phosphorylation which lead to the down-regulation of MITF and tyrosinase expressions. These results suggest that chaetocin could be used to treat various hyperpigmentation disorders and in whitening cosmetics as well. Acknowledgments This research was supported by the Korean Health Technology R&D Project (grant no. A121851). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2015.12.021. References [1] N. Agar, A.R. Young, Melanogenesis: a photoprotective response to DNA damage? Mutat. Res. 571 (2005) 121e132. [2] S. Plensdorf, J. Martinez, Common pigmentation disorders, Am. Fam. Physician 79 (2009) 109e116. [3] M.A. Tucker, Melanoma epidemiology, Hematol. Oncol. Clin. North Am. 23 (2009) 383e395 vii. [4] A. Bonci, A. Patrizi, Pigmentary demarcation lines in pregnancy, Arch. Dermatol. 138 (2002) 127e128. [5] C.B. Lynde, J.N. Kraft, C.W. Lynde, Topical treatments for melasma and postinflammatory hyperpigmentation, Skin Ther. Lett. 11 (2006) 1e6. [6] R. Nussbaum, A.V. Benedetto, Cosmetic aspects of pregnancy, Clin. Dermatol. 24 (2006) 133e141. [7] M. Yoshida, Y. Takahashi, S. Inoue, Histamine induces melanogenesis and morphologic changes by protein kinase A activation via H2 receptors in human normal melanocytes, J. Invest. Dermatol. 114 (2000) 334e342. [8] R. Busca, R. Ballotti, Cyclic AMP a key messenger in the regulation of skin pigmentation, Pigment Cell Res. 13 (2000) 60e69. [9] A. Slominski, M.A. Zmijewski, J. Pawelek, L-tyrosine and L-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions, Pigment Cell Melanoma Res. 25 (2012) 14e27. [10] A. Slominski, J. Wortsman, Neuroendocrinology of the skin, Endocr. Rev. 21 (2000) 457e487. [11] A. Slominski, D.J. Tobin, S. Shibahara, J. Wortsman, Melanin pigmentation in mammalian skin and its hormonal regulation, Physiol. Rev. 84 (2004) 1155e1228. [12] A.T. Slominski, M.A. Zmijewski, C. Skobowiat, B. Zbytek, R.M. Slominski, J.D. Steketee, Sensing the environment: regulation of local and global homeostasis by the skin's neuroendocrine system, Adv. Anat. Embryol. Cell Biol. 212 (2012) 1e115 v, vii. [13] A.T. Slominski, M.A. Zmijewski, B. Zbytek, D.J. Tobin, T.C. Theoharides, J. Rivier, Key role of CRF in the skin stress response system, Endocr. Rev. 34 (2013) 827e884. [14] M. Pucci, N. Pasquariello, N. Battista, M. Di Tommaso, C. Rapino, F. Fezza, M. Zuccolo, R. Jourdain, A. Finazzi Agro, L. Breton, M. Maccarrone, Endocannabinoids stimulate human melanogenesis via type-1 cannabinoid
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receptor, J. Biol. Chem. 287 (2012) 15466e15478. [15] J. Vachtenheim, J. Borovansky, “Transcription physiology” of pigment formation in melanocytes: central role of MITF, Exp. Dermatol. 19 (2010) 617e627. [16] T. Nishimura, T. Kometani, S. Okada, N. Ueno, T. Yamamoto, Inhibitory effects of hydroquinone-alpha-glucoside on melanin synthesis, Yakugaku Zasshi 115 (1995) 626e632. [17] H. Ando, Y. Funasaka, M. Oka, A. Ohashi, M. Furumura, J. Matsunaga, N. Matsunaga, V.J. Hearing, M. Ichihashi, Possible involvement of proteolytic degradation of tyrosinase in the regulatory effect of fatty acids on melanogenesis, J. Lipid Res. 40 (1999) 1312e1316. [18] J.L. O'Donoghue, Hydroquinone and its analogues in dermatology e a riskbenefit viewpoint, J. Cosmet. Dermatol. 5 (2006) 196e203. [19] Z.D. Draelos, Skin lightening preparations and the hydroquinone controversy, Dermatol. Ther. 20 (2007) 308e313. [20] C. Yao, J.H. Oh, I.G. Oh, C.H. Park, J.H. Chung, 6-Shogaol inhibits melanogenesis in B16 mouse melanoma cells through activation of the ERK pathway, Acta Pharmacol. Sin. 34 (2013) 289e294. [21] Y.T. Fu, C.W. Lee, H.H. Ko, F.L. Yen, Extracts of Artocarpus communis decrease alpha-melanocyte stimulating hormone-induced melanogenesis through activation of ERK and JNK signaling pathways, Sci. World J. 2014 (2014) 724314. [22] H.E. Kim, A. Ishihara, S.G. Lee, The effects of caffeoylserotonin on inhibition of melanogenesis through the downregulation of MITF via the reduction of intracellular cAMP and acceleration of ERK activation in B16 murine melanoma cells, BMB Rep. 45 (2012) 724e729. [24] D.S. Kim, Y.M. Jeong, I.K. Park, H.G. Hahn, H.K. Lee, S.B. Kwon, J.H. Jeong, S.J. Yang, U.D. Sohn, K.C. Park, A new 2-imino-1,3-thiazoline derivative, KHG22394, inhibits melanin synthesis in mouse B16 melanoma cells, Biol. Pharm. Bull. 30 (2007) 180e183. [25] J.S. Bae, H.S. Park, J.W. Park, S.H. Li, Y.S. Chun, Red ginseng and 20(S)-Rg3 control testosterone-induced prostate hyperplasia by deregulating androgen receptor signaling, J. Nat. Med. 66 (2012) 476e485. [26] Y.S. Lai, J.Y. Chen, H.J. Tsai, T.Y. Chen, W.C. Hung, The SUV39H1 inhibitor chaetocin induces differentiation and shows synergistic cytotoxicity with other epigenetic drugs in acute myeloid leukemia cells, Blood Cancer J. 5 (2015) e313. [27] A. Lakshmikuttyamma, S.A. Scott, J.F. DeCoteau, C.R. Geyer, Reexpression of epigenetically silenced AML tumor suppressor genes by SUV39H1 inhibition, Oncogene 29 (2010) 576e588. [28] C.R. Isham, J.D. Tibodeau, W. Jin, R. Xu, M.M. Timm, K.C. Bible, Chaetocin: a promising new antimyeloma agent with in vitro and in vivo activity mediated via imposition of oxidative stress, Blood 109 (2007) 2579e2588. [29] J.D. Tibodeau, L.M. Benson, C.R. Isham, W.G. Owen, K.C. Bible, The anticancer agent chaetocin is a competitive substrate and inhibitor of thioredoxin reductase, Antioxid. Redox Signal 11 (2009) 1097e1106. [30] V.J. Hearing, K. Tsukamoto, Enzymatic control of pigmentation in mammals, FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 5 (1991) 2902e2909. [31] H. Ando, H. Kondoh, M. Ichihashi, V.J. Hearing, Approaches to identify inhibitors of melanin biosynthesis via the quality control of tyrosinase, J. Invest. Dermatol. 127 (2007) 751e761. [32] S. Leppa, R. Saffrich, W. Ansorge, D. Bohmann, Differential regulation of c-Jun by ERK and JNK during PC12 cell differentiation, EMBO J. 17 (1998) 4404e4413. [33] S. Cowley, H. Paterson, P. Kemp, C.J. Marshall, Activation of MAP kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells, Cell 77 (1994) 841e852. [34] D.S. Kim, S.Y. Kim, J.H. Chung, K.H. Kim, H.C. Eun, K.C. Park, Delayed ERK activation by ceramide reduces melanin synthesis in human melanocytes, Cell Signal 14 (2002) 779e785. [35] M. Khaled, L. Larribere, K. Bille, J.P. Ortonne, R. Ballotti, C. Bertolotto, Microphthalmia associated transcription factor is a target of the phosphatidylinositol-3-kinase pathway, J. Invest. Dermatol. 121 (2003) 831e836.
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Chaetocin inhibits IBMX-induced melanogenesis in B16F10 mouse melanoma cells through activation of ERK Jung-Soo Bae a, b, c, 1, Mira Han a, b, c, 1, Cheng Yao a, b, c, Jin Ho Chung a, b, c, d, * a
Department of Dermatology, Seoul National University College of Medicine, Seoul, Republic of Korea Laboratory of Cutaneous Aging Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea c Institute of Human-Environment Interface Biology, Seoul National University, Seoul, Republic of Korea d Institute on Aging, Seoul National University, Seoul, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2015 Received in revised form 22 December 2015 Accepted 29 December 2015 Available online 31 December 2015
Chaetocin is a natural product isolated from Chaetomium species that has anti-bacterial and antimyeloma activities. In this study, we investigated the inhibitory effect of chaetocin on melanogenesis and the underlying mechanisms in B16F10 mouse melanoma cells. In the present study, chaetocin significantly inhibited IBMX-induced melanin production and tyrosinase activity without any cytotoxicity. Furthermore, chaetocin down-regulated both the protein and mRNA levels of tyrosinase, which is a specific enzyme that catalyzes the conversion of tyrosine to melanin. We also observed that the protein level of MITF was significantly reduced by chaetocin treatment. In addition, we found that the antimelanogenic effect of chaetocin was suppressed by treatment with the specific ERK inhibitor (PD98059). Accordingly, chaetocin inhibited melanogenesis via suppressing the protein level of MITF followed by activation of the ERK signaling pathway. These data suggest that chaetocin may be a potential anti-melanogenic agent for use in skin-whitening cosmetics and a topical agent for treatment of hyperpigmentation disorders. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Chaetocin Melanin Tyrosinase Microphthalmia-associated transcription factor Extracellular signal-regulated kinases ERK
1. Introduction Melanin is the major pigment synthesized by epidermal melanocytes in the skin and it determines the skin color due to its own color: eumelanin (black) and pheomelanin (yellow). In addition, melanin has an important role in the physiological defense of skin protection from ultraviolet (UV) radiation. However, repetitive UV irradiation induces increased accumulation of melanin and leads to skin hyperpigmentation [1]. Hyperpigmented skin has an unaesthetic appearance such as chloasma and freckles [2,3]. The biosynthesis of melanin is induced by several stimuli such
Abbreviations: a-MSH, a-melanocyte-stimulating hormone; PBS, phosphatebuffered saline; DMEM, Dulbecco's modified Eagle's media; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IBMX, 3-isobutyl-1methylxanthine; MITF, microphthalmia-associated transcription factor; UV, ultraviolet; L-DOPA, L-3,4-dihydroxyphenylalanine; TRP, tyrosinase-related protein; ERK, extracellular signal-regulated kinases. * Corresponding author. Department of Dermatology, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Republic of Korea. E-mail address: [email protected] (J.H. Chung). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.cbi.2015.12.021 0009-2797/© 2015 Elsevier Ireland Ltd. All rights reserved.
as UV radiation, inflammation, hormones, or other skin injuries [4e6]. For instance, inflammation is closely related with melanogenesis, and a large number of inflammatory cytokines have been reported as melanogens. Histamine, an ubiquitous inflammatory mediator, is a representative of melanogen induced by inflammation [7]. In addition, several chemicals elevating intracellular cAMP (forskolin and IBMX), and substrates of melanogenesis such as Ltyrosine and L-dihydroxyphenylalanine (L-DOPA) can also activate melanogenesis [8,9]. Moreover, UV radiation is considered as the primary factor of melanogenesis in normal physiological conditions. UV radiation stimulates cutaneous hypothalamic-pituitaryadrenal (HPA) axis to regulate local stress responses, and produces POMC peptides such as MSH, b-endorphin and ACTH. These peptides increase melanin pigmentation by interacting with melanocortin receptor on melanocytes [10e13]. Endocannabinoids are also generated in keratinocytes in response to UV radiation, and that increases melanin synthesis by activating endocannabinoid signaling pathway [14]. In general, these melanogenic stimuli lead to an increase in microphthalmia-associated transcription factor (MITF) expression. MITF is the master regulator of melanin synthesis which induces the transcription of melanogenic specific enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1),
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and dopachrome tautomerase (DCT) [15]. Therefore, downregulation of these molecules involved in the process of melanin synthesis is regarded as a way of skin whitening. There are various skin whitening agents, such as kojic acid, arbutin, and linoleic acid. Kojic acid and arbutin are widely used in Northeast Asia as cosmetic ingredients due to their anti-tyrosinase activity. Arbutin is a glycosylated hydroquinone extracted from the bearberry plant, and it has generally been used as a treatment for hyperpigmentation disorders in the past [16]. In addition, linoleic acid suppresses tyrosinase activity by accelerating ubiquitindependent degradation of tyrosinase protein [17]. However, some of these agents have been reported to cause skin irritation. It has been reported that kojic acid causes skin irritation and side effects such as cytotoxicity, dermatitis, and skin cancer [8]. Arbutin has been also banned due to its side effects involving exogenous ochronosis and perdurable depigmentation [18,19]. For these reasons, some countries have placed restrictions on the use of these chemicals as cosmetic ingredients. Recently, numerous reports have focused on the development of a safe skin-whitening agent from natural sources due to side effects of the established agents. 6-Shogaol, one of the ingredients present in ginger, decreases melanogenesis by activation of the ERK pathway [20]. Extracts of Artocarpus communis also alleviate aMSH-stimulated melanin accumulation in B16F10 melanoma cells [21]. In addition, Caffeoylserotonin, one of serotonin derivatives from a wide range of plant species inhibits melanogenesis [22]. Chaetocin is one of the natural products isolated from Chaetomium species fungi with antimicrobial and cytostatic activities. However, its pharmacological action on the skin has not been fully understood. In this study, we investigated the anti-melanogenic effect of chaetocin on IBMX-induced melanogenesis in B16F10 mouse melanoma cells as well as explored the underlying molecular mechanisms involved in this process. 2. Materials and methods 2.1. Chemicals and reagents Chaetocin, 3-isobutyl-1-methylxanthine (IBMX), L-DOPA, and 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). The inhibitor PD98059 was obtained from Calbiochem (San Diego, CA, USA). Specific antibodies for phospho-ERK1/2, total ERK1/2, and phospho-AKT were purchased from Cell Signaling (Beverly, MA, USA). Antibodies recognizing tyrosinase (C-19) and actin (I-19) were obtained from Santa Cruz (Santa Cruz, CA, USA). MITF antibody (MS-771-P0) was purchased from NeoMarkers (Fremont, CA, USA). 2.2. Cell culture B16F10 mouse melanoma cell line obtained from the Korean Cell Line Bank (Seoul, Korea) was cultured in Dulbecco's modified Eagle's media (DMEM; Gibco-BRL, Carlsbad, CA, USA) without phenol red, supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) and penicillin/streptomycin (400 U/mL, 50 g/L) at 37 C under the humidified condition with 5% CO2. To maintain cell characteristics, cells were split every 2 days. 2.3. Cell viability assay B16F10 cell viability was determined using MTT-based assay. B16F10 cells (2.5 103 cells/well) were seeded into 96-well plates and incubated with chaetocin (10, 20, 30, or 40 nM) for 24 h and 48 h, respectively. After incubation, culture plates were treated
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with MTT (dissolved in PBS to 0.5 g/L) for 4 h. The supernatant was removed and the formazan crystals were dissolved in DMSO. The amount of formazan was quantified at 570 nm using ELISA reader (Thermo Fisher Scientific Inc., USA). 2.4. Measurement of melanin content Measurement of intracellular melanin content was quantified by using the previously described method with slight modification [20]. B16F10 cells were stimulated by IBMX and incubated with chaetocin or the inhibitor PD98059 at the same time for 48 h. The cell pellets were harvested and then dissolved in 1 N NaOH containing 10% DMSO at 80 C for 1 h. The melanin content was analyzed at 475 nm using ELISA reader. For accurate calculation of melanin content, each level of melanin was normalized to the protein content. 2.5. Tyrosinase activity Tyrosinase activity was determined using the method described previously [24]. B16F10 cells were first treated with IBMX and further treated with serial concentrations of chaetocin at the same time. After 48 h incubation, the cells were washed with cold PBS and lysed in phosphate saline (pH 6.8) containing Triton-X100 (1.0%) and protease inhibitor cocktail (Roche Diagnostics). Cell lysates were centrifuged and then the protein content of the supernatant was normalized using the Bradford protein assay. Then, 90 mL of cell extract was placed in a 98-well plate treated with freshly prepared 10 mL of L-DOPA (final concentration of 1 mmol/L) in 25 mM phosphate buffer (pH 6.8) and incubated at 37 C for 20 min. Absorbance was measured at 475 nm using an ELISA reader. 2.6. Western blot analysis B16F10 cells (2.5 105 cells) were seeded into 60 mm dishes and incubated with chaetocin. Cells were washed with cold PBS and homogenized in 50 mM TriseHCl (pH 7.4) buffer containing 1% NP40, 0.25% deoxycholic acid, 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma Aldrich). Protein contents of total cell lysates were determined using BCA protein assay (Sigma Aldrich). Each sample was adjusted to same concentration and 20 mg of protein per lane was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a PVDF membrane. The membrane was blocked with 5% fat-free milk in TBST (Tris-buffered saline containing 0.4% Tween 20) followed by incubation with appropriate primary antibodies. After 24 h incubation with primary antibodies, blots were further incubated for 1 h with horseradish peroxidase-conjugated secondary antibody. Immune complexes were visualized using an enhanced chemiluminescence detection system (GE Healthcare). 2.7. Reverse transcription and real-time quantitative PCR Total RNA was isolated from B16F10 cells using RNAiso Plus (Takara Bio Inc., Shiga, Japan), as previously described [25]. Isolated RNAs were reverse-transcribed into cDNAs using the First Strand cDNA Synthesis Kit (MBI Fermentas, Vilnius, Lithuania). The synthesized cDNAs were subjected to real-time quantitative PCR and quantitation of PCR products was performed by 7500 Real time PCR System (Applied Biosystems, Foster City, CA) with the SYBR Premix Ex Taq II kit (Takara Bio). The PCR reactions were performed with the primers for MITF, tyrosinase, TRP-1, and endogenous reference 36B4. Data were analyzed using the 2DDCT method and data are expressed as the fold number of gene expression.
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2.8. Statistical analyses All data were analyzed using Microsoft Excel 2007 software. Results are expressed as means ± SEM, and statistical analyses were performed using a Student's t-test. P-values of less than 0.05 were considered statistically significant.
decreased the expression levels of tyrosinase protein (Fig. 2D) and mRNA (Fig. 2E). In addition, we observed that the level of TRP-1 mRNA was decreased by chaetocin treatment (Fig. 2F). These results indicate that chaetocin has an anti-melanogenic effect on B16F10 mouse melanoma cells. 3.3. Chaetocin inhibits MITF protein expression
3. Results 3.1. Cytotoxicity of chaetocin in B16F10 mouse melanoma cells To investigate whether chaetocin has cytotoxic effects, we treated B16F10 mouse melanoma cells with various doses of chaetocin in the absence or presence of IBMX. The viability of B16F10 cells was measured using the MTT-based assay. The cell viability was measured at 48 h after treatment with 10, 20, 30, or 40 nM chaetocin. We observed that chaetocin had no significant effect on cell viability either in the absence (Fig. 1A) or presence (Fig. 1B) of IBMX. The results showed that chaetocin has little cytotoxic effect in B16F10 cells at the concentrations used in the present study. 3.2. Chaetocin inhibits IBMX-induced melanin synthesis in B16F10 mouse melanoma cells We next investigated the effect of chaetocin on IBMX-induced melanogenesis. We demonstrated that chaetocin inhibited IBMXinduced melanin synthesis in a dose-dependent manner (Fig. 2A, B). Tyrosinase is a well-known catalyzing enzyme which mediates melanin production. Therefore, we investigated the effects of chaetocin on tyrosinase enzymatic activity and tyrosinase expression after chaetocin treatment. Chaetocin significantly decreased the IBMX-induced tyrosinase activity in a dose-dependent manner (Fig. 2C). We also observed that treatment with chaetocin
To understand the transcriptional regulation of tyrosinase and TRP-1 mRNA expression, we investigated the effect of chaetocin on the expression of MITF, which is a major transcription factor for tyrosinase and TRP-1. After treatment with chaetocin, the protein level of MITF was down-regulated in a dose-dependent manner (Fig. 3A). Chaetocin treatment also decreased the IBMX-induced MITF protein expression in a time-dependent manner (Fig. 3B). These results indicate that chaetocin has inhibitory activity on MITF protein expression. 3.4. Suppression of the ERK pathway relieved chaetocin-induced inhibition of melanogenesis To understand how chaetocin regulates MITF expression, we investigated the time-dependent effects of chaetocin on MITF expression and phosphorylation of ERK and AKT. We confirmed that chaetocin decreased the IBMX-induced MITF protein expression (Fig. 4A). On the other hand, chaetocin increased the phosphorylation of ERK, but not of AKT. To investigate whether increased phosphorylation of ERK is involved in inhibition of MITF production, pretreatment with a selective inhibitor of the ERK pathway, PD98059, was performed before chaetocin treatment. We found that treatment with PD98059 relieved chaetocin-induced inhibition of MITF expression. Phosphorylation of ERK induced by chaetocin was also inhibited by PD98059 (Fig. 4B). To further investigate the effect of chaetocin-induced ERK phosphorylation on
Fig. 1. Effects of chaetocin on cell viability in B16F10 mouse melanoma cells. Cell viability was analyzed by MTT assay. B16F10 cells were treated for 48 h with (A) chaetocin at a concentration of 10, 20, 30, or 40 nM and (B) chaetocin plus IBMX (100 mM). (C) The chemical structure of chaetocin. Each bar represents the mean ± SEM of three independent experiments.
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Fig. 2. Effect of chaetocin on IBMX-induced melanin production and tyrosinase activity. B16F10 mouse melanoma cells were co-cultured with chaetocin (10, 20, or 30 nM) and IBMX (100 mM). After incubation for 48 h, the melanin content was analyzed. (A) Photograph of cell culture dishes. (B) Melanin content results are presented as the number of folds. (C) Tyrosinase activity was evaluated by L-DOPA oxidation assay. Tyrosinase protein expression was determined by Western blotting at 24 h after treatment (D). mRNA levels of tyrosinase (E) and TRP-1 (F) were determined by RT-qPCR at 12 h after treatment. All RT-qPCR experiments were performed five times. Each bar represents the mean ± SEM. Asterisks in all graphs denote a value that is statistically different (*, P < 0.05; **, P < 0.01) between the IBMX only and IBMX þ chaetocin treated groups.
Fig. 3. Chaetocin inhibits MITF protein expression without affecting its mRNA level. B16F10 cells were treated with 100 mM IBMX or/and chaetocin (10, 20, or 30 nM) (A) MITF protein expression was measured at 4 h after various doses of chaetocin treatment. (B) MITF protein expression was also checked at different time points after co-treatment with chaetocin (30 nM) and IBMX (100 mM). Protein expression was determined by Western blotting.
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Fig. 4. Suppression of the ERK pathway relieved chaetocin-induced inhibition of melanogenesis. (A) Cells were incubated with IBMX (100 mM) plus chaetocin (30 nM) for the indicated times mentioned above. Changes in MITF, p-AKT, and p-ERK protein expressions were visualized by Western blotting. (B) B16F10 cells were treated with IBMX alone or IBMX plus 30 nM chaetocin in the absence or presence of PD98059 (20 mM). Changes in MITF and phosphorylated ERK expressions were checked at 4 h after treatment. Melanin contents were measured at 48 h after treatment. All results are indicated as the mean ± SEM of four different experiments. Asterisks in the graph denote a value that is statistically different (* and #, P < 0.05).
melanin production, melanin contents were evaluated in the presence of PD98059 in chaetocin-treated cells. The level of melanin inhibited by chaetocin was increased by PD98059 treatment (Fig. 4C). Thus, these results indicated that activation of the ERK pathway is involved in chaetocin-induced reduction of melanogenesis. 4. Discussion Chaetocin is a natural product isolated from Chaetomium species fungi, and it is known as a specific inhibitor of the lysine-specific histone methyltransferase SUV39H1 (suppressor of variegation 3e9 homolog 1) in Drosophila melanogaster [18,19]. Other reports have shown that chaetocin reduces the methylation of H3K9 and H3K27 [26,27]. Chaetocin has potent anti-myeloma activity which is attributable to induction of reactive oxygen species (ROS) imposed by inhibition of thioredoxin reductase [28,29]. However, in spite of many published reports, the effect of chaetocin on melanogenesis has not been studied well. In the present study, we investigated the effect of chaetocin on melanin synthesis and the underlying molecular mechanisms involved in this process in B16F10 mouse melanoma cells. Tyrosinase, a copper-containing enzyme, is an important enzyme for controlling the production of melanin [30]. Hence, molecules controlling tyrosinase expression and activity have been considered as treatment of hyperpigmentation [31]. In this study,
we showed the anti-melanogenic effect of chaetocin in a dosedependent manner without any cytotoxicity in B16F10 mouse melanoma cells. Furthermore, we found that tyrosinase expression and activity were decreased by chaetocin treatment in B16F10 mouse melanoma cells. MITF is a transcription factor that is closely related to the development of melanocytes and osteoclasts. As a major transcription factor in melanogenesis, MITF can control the expression of several genes which act as critical enzymes in the conversion of tyrosine to melanin. In this study, we demonstrated that chaetocin dramatically decreased MITF protein expression. The MAPK/ERK pathway plays an important role in proliferation and differentiation of various types of cells [32,33]. Especially in melanocytes, the ERK pathway can control melanogenesis by regulating MITF expression. Several reports have noted that phosphorylation of ERK attenuated a-MSH and IBMX-induced MITF protein expression at the posttranslational level [27]. Previous reports have demonstrated that the activated ERK induces the phosphorylation of MITF at serine-73 and leads to MITF ubiquitin-dependent degradation [15,34]. The PI3K/Akt signaling pathway also has an important role in melanin synthesis [35]. Activation of PI3K/Akt pathway reduces melanogenesis by regulating tyrosinase transcription in melanocyte. Therefore, we studied the effect of chaetocin on the Akt activation, but observed no significant changes in Akt phosphorylation. On the other hand, chaetocin effectively activated the ERK signaling pathway, and lead to down-regulation of MITF expression.
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Moreover, down-regulated MITF protein levels by chaetocin were restored by inhibiting ERK activation by PD98059. In addition, the inhibitory effect of chaetocin against melanin synthesis was alleviated by inhibiting the ERK pathway. In summary, we identified that chaetocin inhibited melanogenesis in IBMX-treated B16F10 cells without any cytotoxicity. We found that chaetocin induced ERK phosphorylation which lead to the down-regulation of MITF and tyrosinase expressions. These results suggest that chaetocin could be used to treat various hyperpigmentation disorders and in whitening cosmetics as well. Acknowledgments This research was supported by the Korean Health Technology R&D Project (grant no. A121851). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2015.12.021. References [1] N. Agar, A.R. Young, Melanogenesis: a photoprotective response to DNA damage? Mutat. Res. 571 (2005) 121e132. [2] S. Plensdorf, J. Martinez, Common pigmentation disorders, Am. Fam. Physician 79 (2009) 109e116. [3] M.A. Tucker, Melanoma epidemiology, Hematol. Oncol. Clin. North Am. 23 (2009) 383e395 vii. [4] A. Bonci, A. Patrizi, Pigmentary demarcation lines in pregnancy, Arch. Dermatol. 138 (2002) 127e128. [5] C.B. Lynde, J.N. Kraft, C.W. Lynde, Topical treatments for melasma and postinflammatory hyperpigmentation, Skin Ther. Lett. 11 (2006) 1e6. [6] R. Nussbaum, A.V. Benedetto, Cosmetic aspects of pregnancy, Clin. Dermatol. 24 (2006) 133e141. [7] M. Yoshida, Y. Takahashi, S. Inoue, Histamine induces melanogenesis and morphologic changes by protein kinase A activation via H2 receptors in human normal melanocytes, J. Invest. Dermatol. 114 (2000) 334e342. [8] R. Busca, R. Ballotti, Cyclic AMP a key messenger in the regulation of skin pigmentation, Pigment Cell Res. 13 (2000) 60e69. [9] A. Slominski, M.A. Zmijewski, J. Pawelek, L-tyrosine and L-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions, Pigment Cell Melanoma Res. 25 (2012) 14e27. [10] A. Slominski, J. Wortsman, Neuroendocrinology of the skin, Endocr. Rev. 21 (2000) 457e487. [11] A. Slominski, D.J. Tobin, S. Shibahara, J. Wortsman, Melanin pigmentation in mammalian skin and its hormonal regulation, Physiol. Rev. 84 (2004) 1155e1228. [12] A.T. Slominski, M.A. Zmijewski, C. Skobowiat, B. Zbytek, R.M. Slominski, J.D. Steketee, Sensing the environment: regulation of local and global homeostasis by the skin's neuroendocrine system, Adv. Anat. Embryol. Cell Biol. 212 (2012) 1e115 v, vii. [13] A.T. Slominski, M.A. Zmijewski, B. Zbytek, D.J. Tobin, T.C. Theoharides, J. Rivier, Key role of CRF in the skin stress response system, Endocr. Rev. 34 (2013) 827e884. [14] M. Pucci, N. Pasquariello, N. Battista, M. Di Tommaso, C. Rapino, F. Fezza, M. Zuccolo, R. Jourdain, A. Finazzi Agro, L. Breton, M. Maccarrone, Endocannabinoids stimulate human melanogenesis via type-1 cannabinoid
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