through the MEK/ERK signaling pathway
Wen-Sheng Liu1, Yu-Diao Kuan2, Kuo-Hsun Chiu3, Wei-Kuang Wang4 Fu-Hsin Chang1, Chen-Hsun Liu1,5 and Che-Hsin Lee 2, 6,*
1 Asia-Pacific Biotech Developing, Inc. Kaohsiung, 806, Taiwan
2 Graduate Institute of Basic Medical Science, School of Medicine, China Medical University,
Taichung 404, Taiwan
3 Department and Graduate Institute of Aquaculture, National Kaohsiung Marine University,
Kaohsiung 811, Taiwan
4 Department of Environmental Engineering and Science, Feng Chia University, Taichung 407,
Taiwan
5 Department of Biological Science and Technology of I-Shou University, Kaohsiung 840, Taiwan 6 Department of Microbiology, School of Medicine, China Medical University, Taichung 404,
Taiwan
* Author to whom correspondence should be addressed; E-Mails: [email protected] (C.-H.L.). Abstract: Reducing hyperpigmentation has been a big issue for years. Even though
pigmentation is a normal mechanism protecting skin from UV-causing DNA damage and oxidative stress; it’s still an aesthetic problem for many people. Bacteria can produce some compounds in response to their environment. These compounds are widely used in cosmetic and pharmaceutical applications. Some probiotics have immunomodulatory activities and modulate the symptoms of several diseases. Previously, we found that the extracts of Rhodobacter sphaeroides (LycogenTM) inhibited nitric oxide production and
inducible nitric-oxide synthase expression in activated macrophages. In this study, we sought to investigate an anti-melanogenic signaling pathway in α-melanocyte stimulating hormone (α-MSH)-treated B16F10 melanoma cells and zebrafish. Treatment with LycogenTM inhibited the cellular melanin contents and expression of melanogenesis-related
protein, including microthphalmia associated transcription factor (MITF) and tyrosinase in B16F10 cells. Moreover, LycogenTM reduced phosphorylation of MEK/ERK without
affecting phosphorylation of p38. Meanwhile, LycogenTM decreased zebrafish melanin
expression in dose-dependent manner. These findings establish that LycogenTM as a new
target in melanogenesis and suggest a mechanism of action through the ERK signal pathway. Our results suggested that LycogenTM may have potential cosmetic usage in the
future.
Keywords: Rhodobacter sphaeroides; LycogenTM; melanogenesis
1. Introduction
Melanin plays important photo-protective roles in the carcinogenic and deleterious effects of ultraviolet radiation of solar light. Abnormal melanogenesis is a feature of many human skin diseases 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
including pigmentary disorders and melanoma. In European, skin lighterers are applied for the prevention and treatment of irregular hyperpigmentation, such as melasma and age spots. Past studies focused on the screening of nature compounds from plant sources for anti-melanogenic agent [1, 2]. In melanocytes, α-melanocytes stimulating hormone (α-MSH) through the binding to the melanocortin-1 receptor (MC1R) and up-regulation of cAMP pathway induces melanin synthesis. At least three enzymes are required for melanin synthesis including tyrosinase, tyrosinase-related protein 1(TRP1), and tyrosinase-related protein 2 (TRP2), through a transcriptional mechanism involving microphthalmia-associated transcription factor (MITF), the master gene of melanocyte differentiation. Some of the agents with anti-oxidative activity have been shown to play an important role in the inhibition of melanogenesis. Bacteria can produce some compounds in response to their environment. These compounds are widely used in cosmetic and pharmaceutical applications. Some probiotics have immunomodulatory activities and modulate the symptoms of several diseases. Previously, we found that the extracts of Rhodobacter sphaeroides (LycogenTM) inhibited nitric oxide production and
inducible nitric-oxide synthase expression in activated macrophages [3]. Meanwhile, the effect of LycogenTM, a potent anti-inflammatory agent, was evaluated in mice with dextran sodium sulfate
(DSS)-induced colitis. Oral administration of LycogenTM reduced the expressions of proinflammatory
cytokines (tumor necrosis factor-α and interleukin-1β) in mice [4]. In this study, we sought to investigate an anti-melanogenic signaling pathway in α-MSH-treated B16F10 melanoma cells and zebrafish.
2. Results
2.1 Evaluation of anti-melanogenic activity of LycogenTM in vitro
In this study, we used mouse B16F10 melanoma cell to evaluate the anti-melanogenic activity of LycogenTM. The results for in vitro treatment of B16F10 cells with LycogenTM for cell survival, and
melanin content are shown in Figure 1. Thus dose (6.25 μM-12.5μM ) without significant cytotoxicity were chose to determine the effects of LycogenTM on melanin production (Figure 1a). The melanin
content of B16 cells increased considerably after stimulation by α-MSH. Treatment with LycogenTM
resulted in a significant and dose-dependent decrease in the melanin content of α-MSH-stimulated B16F10 cells (Figure 1b). Taken together, these results suggest that LycogenTM influenced the
melanogenesis in B16F10 cells.
Figure 1. Effects of LycogenTM on cell viability and melanin production in B16F10 cell. B16F10 cells were treated with indicated concentrations of LycogenTM for 48 h. (a)
Cell viability was measured by WST-1 assay. (b) Effect of LycogenTM on cellular melanin
content.＊
,
p＜0.05;＊＊,
p＜0.01; ＊＊＊,
p＜0.001. (mean ± SD, n = 6). Each experiment was repeated three times with similar results.
2.2. LycogenTM dose-dependently down-regulated the expression levels of tyrosinase and MITF 2 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74
To elucidate the mechanisms underlying the anti-melanogenic activity of LycogenTM, we fist
examined the expression levels of tyrosinase by immunoblotting. As shown in Figure 2, LycogenTM
dose-dependently reduced the expression of tyrosinase. Tyrosinase are transcriptionally regulated by MITF. Interesting, we found that LycogenTM dose-dependently inhibited MITF expression.
Figure 2. The expression levels of tyrosinase and MITF after LycogenTM treatment.
B16F10 cells were treated with LycogenTM at the concentration of 6.25, 12.5 or 25 μM for
48 h. The protein expression was determined by immunoblotting. Inserted values indicated relative proteins expression in comparison with β-actin. Each experiment was repeated three times with similar results.
2.3 Effects of LycogenTM on the mitogen-activated protein kinase (MAPKs) signaling pathway
MAPK kinase including ERK and p38 plays an important role in melanogenesis [5, 6]. Up-regulation of ERK signaling is related to the down-regulation of melanin synthesis. However, phosphorylation of p38 can up-regulate the MITF expression. We examined the influence of LycogenTM on the signaling
pathway of p38 and ERK in an attempt to further understand the molecular mechanism involved in the anti-melanogenic activity of LycogenTM by immunoblotting. In this study, we found that α-MSH
significant reduced the ERK signaling pathway (Figure 3). However, LycogenTM reversed the
phenomenon. LycogenTM dramatically increased the ERK signaling pathway. Meanwhile, p38
signaling pathway was not influenced by LycogenTM. Furthermore, the α-MSH-induced response of
MAPK/ERK pathway associated factors, ERK and MEK were determined (Figure 4a). However, MEK and ERK phosphorylation significantly decreased after α-MSH treatment. In order to investigate the relationship between melanogenesis and the ERK pathway, B16F10 cells were treated with inhibitor PD98059 before LycogenTM treatment. PD98059 is a potent and selective inhibitor of MAP
kinase kinase (also known as MAPK/ERK kinase or MEK kinase). It mediates its inhibitory properties by binding to the ERK-specific MAP kinase MEK, therefore preventing phosphorylation of ERK1/2 (p44/p42 MAPK) by MEK. Immunblot analysis showed that PD98059 reduced the expression of ERK phosphorylation. The content of melanin also dramatically increased after PD98059 treatment (Figure 5b). LycogenTM did not reverse melanogenesis by PD98059. The similar results were also observed in
B16F10 cells after ERK dominant negative plasmids transduction (Figure 4c). Consistent with PD98059 treatment, ERK dominant negative plasmid abolished the inhibitory effect of LycogenTM on
melanin synthesis. The ERK signaling inhibitors blocked the hypopigmenting effects induced by LycogenTM. These results suggested that LycogenTM increased MEK/ERK cascade promoting MITF
degradation.
Figure 3. Effect of LycogenTM on the expression levels of MAPKs and phosphorylated-MAPKs in cells. B16F10 cells were treated with LycogenTM at the concentration of 6.25,
12.5 or 25 μM for 48 h. The protein expression was determined by immunoblot analysis.
The expression of β-actin served as the quantitative control. Inserted values indicated relative proteins expression in comparison with β-actin. Each experiment was repeated three times with similar results.
5 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112
Figure 4. Lycogen reduces melanin content through ERK pathway. B16F10 cells were treated
with LycogenTM at the concentration of 12.5 μM for 48 h and PD98059 (50 μM) for 24 h. (a) The
protein and (b) melanin expression were measured. The expression of β-actin served as the quantitative control. Inserted values indicated relative proteins expression in comparison with β-actin. (c) The melanin was detected in B16F10 cells transiently transfected with ERK dominant negative or with control vector after treatment with LycogenTM.
2.5. Evaluation of anti-melanogenic activity of LycogenTM in vivo
Zebrafishes have been established as a new in vivo model for evaluating the depigmenting activity of melanogenic regulatory compounds. No significant toxicity of LycogenTM was observed in zebrafish
embryos at the tested concentrations up to 100μM (Figure 5a). However, treatment of the embryos with LycogenTM during 48 h significantly reduced the skin melanin in the developed larvae (Figure 5a).
The melanin content of LycogenTM-treated zebrafish was dose dependently decreased compared to that
of untreated fish (Figure 5b).To understand the postulated signal mechanism involved in the inhibitory effect of LycogenTM on melanin synthesis in zebrafish, zebrafish embryos were treated with 12.5μM
for 48 h. The protein expression of p38, ERK and AKT were determined by western blotting. The treatment of LycogenTM led a slight increase in phosphorylated-ERK and phosphorylated-AKT.
Consistent with B16F10 cells, the expression of phosphorylated-p38 was not influenced after LycogenTM treatment. The results point out that LycogenTM inhibited the melanogenesis in vivo.
Figure 5. Effects of LycogenTM on the pigmentation of zebrafish. (a) Image of zebrafish
treated with LycogenTM.(b) Synchronized embryos were treated with various concentration
LycogenTM for 48 h. Melanin pigment was determined by a photometric methods. (mean ±
SD, n = 3). ＊＊
,
p＜0.01; ＊＊＊,
p＜0.001. (c) The protein expression after LycogenTM(12.5μM) treatment were measured by immunoblotting in zebrafish. The expression of total p38, ERK, or AKT served as the quantitative control. Inserted values indicated relative proteins expression in comparison with total protein. Each experiment was repeated three times with similar results.
3. Discussion
We had found that LycogenTM could reduce the melanin production in vitro and in vivo. Although
B16F10 cells expressed MITF, it was reduced after treatment with LycogenTM, as assessed by western
blot analysis in a dose-dependent manner. Herein, we want to point out that LycogenTM could inhibit
melanin production by targeting to ERK signaling pathway. LycogenTM is able to serve as a potential
melanogenesis inhibitor. The suppressed activation of cAMP response element-binding protein (CREB) via the down-regulation of p38 had been reported in hypopigmentation [6]. Herein, we found 8 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148
that LycogenTM did not influence the phosphorylation of p38. It was reported that the activation of
ERK resulted in the phosphorylation of MITF at serine 73, which induced the subsequent ubiquitin-dependent proteasomal degradation [7]. Meanwhile, in the present study, the involvement of ERK signaling pathway was confirmed by the treatment of PD98059 or ERK dominent negative plasmid. A similar anti-melanogenic effect was also described in that c-phycocyanin raised the level of ERK phosphorylation to inhibit the biosynthesis of melanin [7]. These observation might infer that the constitutes of LycogenTM could function as an ERK activator to regulate melanin synthesis. These
results showed that ERK phosphorlation is significantly up-regulated when B16F10 cells and zebrafish are treated with LycogenTM, which suggests that LycogenTM induces hypopigmentation by increasing
ERK phosphorylation in vitro and in vivo. The activation of AKT is responsible for supression for melanin synthesis in melanoma cells and that specific inhibition of the AKT pathway stimulates melanin synthesis. In the present study, zebrafishes treated with LycogenTM also increased the
expression of phosphorylated-AKT. Thus, our results indicate that LycogenTM-induced melanin
reduction may be mediated by the ERK and AKT signaling pathway. Lycogen™ contains ζ-carotene, neurosporene, spheroidenone and methoxyneurosporene accordinh to nuclear magnetic resonance spectroscopy analysis. ζ-Carotene is the precursor of neurosporene, which in turn is the precursor of lycopene [8]. Lycopene, as an anti-oxidative agent, prevents the production of melanin [9]. Meanwhile, neurosporene itself has the ability to protect irradiation with UV-B [10]. Lycogen™ inhibited NO production and iNOS expression in activated macrophage [3], and was capable of improving colonic damage in DSS-induced colitis [4]. Further work is warranted to elucidate the active ingredient(s) in Lycogen™ for hypopigmentation. These findings point out that Lycogen™ might contribute to its therapeutic effect on anti-melanogenesis.
4. Experimental Section
4.1 LycogenTM, cells, mice and zebrafish
R. sphaeroides (WL-APD911) was isolated from mutants using chemical mutagenesis (Bioresource Collection and Research (BCRC), Hsinchu, Taiwan). The R. sphaeroides was cultured in broth. After harvesting, the bacterial broth was centrifuged and washed with ethanol. The bacterial residue is extracted with acetone and then centrifuged by 7,500 rpm for 5 min. The supernatant is filtered through filter paper and a 0.2 μm filter into a round-bottomed flask. The color of the final supernatant is dark red. Acetone is removed completely in oven at 55 °C. The extract of R. sphaeroides was named Lycogen™. Lycogen™ are available from Asia-Pacific Biotech Developing, Inc. (Kaohsiung, Taiwan). Murine B16F10 cells are cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1% glutamine, and 50 μg/ml gentamicin at 37℃ in 5% CO2 [11]. Adult zebrafishes
were obtained from a commercial dealer and 10–15 fishes were kept in 5 l acrylic tank with the following conditions; 28.5oC, with a 14/10 h light/dark cycle. Zebrafishes were fed three times a day, 6
d/week, with flake food supplemented with live brine shrimps (Artemia salina). Embryos were obtained from natural spawning that is induced at the morning by turning on the light. Collection of embryos were completed within 30 min. The experimental protocol adhered to the rules of the Animal 11 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
Protection Act of Taiwan, and was approved by the Laboratory Animal Care and Use Committee of the China Medical University.
4.2 Determinations of anti-melanogenic activity in zebrafish
Synchronized embryos were collected and arrayed by pipette, three to four embryos per well, in 96-well plates containing embryo medium. LycogenTM were dissolved in water, then added to the embryo
medium for 48 h. The effects on the pigmentation of zebrafish were observed under the stereomicroscope. For observation, embryos were dechorionated by forceps, anesthetized in tricaine methanesulfonate solution (Sigma-Aldrich, St. Louis, MO, USA), mounted in 3% methyl cellulose on a depression slide, and photographed under the stereomicroscope MZ16 (Leica Microsystems, Ernst-Leitz-Strasse, Germany). The signals were quantified with ImageJ software (rsbweb.nih.gov/ij/ ). 4.3 Assay of cell proliferation
Cells (105/well) were treated with various concentration of LycogenTM in culture medium for 48 h.
The medium were removed, washed, and replenished with fresh medium supplemented with 2% FBS. Cell proliferation was assessed by the colorimetric WST-1 assay (Dojindo Labs, Tokyo, Japan) according to the manufacturer’s instructions [13].
4.4 Determination of melanin content
At the end of cell culture, the cells were harvested and washed twice with PBS. The pelleted cells were lysed in cold lysis buffer(20 mM sodium phosphate pH 6.8, 1% Triton X-100, 1 mM PMSF, and 1 mM EDTA). After centrifugation at 15 000 g for 15 min, the melanin pellets were dissolved in Soluene-350 (Perkin-Elmer, Waltham, MA, USA) for 15 min at 100oC. The absorbance at 500 nm was
measured [14]. The protein content in each sample was determined by bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL, USA). The working concentrations of 20 μM PD98059 (Sigma-Aldrich). Cells were pretreated inhibitors for 1 h, then LycogenTM was added to cells.
LycogenTM-treated or untreated cells were lysed in cold lysis buffer (20 mM sodium phosphate pH 6.8,
1% Triton X-100, and 1 mM PMSF). 4.5 Western blot analysis
To inhibit ERK signal pathway, cells were pretreated PD98059 for 1 h, then LycogenTM was added to
cells for 48 h. The B16F10 cells were transfected with ERK dominant negative plasmids by lipofectamine 2000 (Invitrogene, Carlsbad, CA, USA). The ERK dominant negative mutant was a gift from Dr. M. Cobb (South-Western Medical Center, Dallas, TX, USA). Cell lysates were prepared by extracting proteins with lysis buffer. Proteins from total cell extracts were fractionated on SDS-PAGE, transferred onto Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham, Little Chalfont, UK), and probed with primary antibodies against tyrosinase (Santa Cruz Biotechnology, Santa Cruz, CA, USA), MITF (Santa Cruz Biotechnology), ERK (Abcam, Cambridge, UK) , 14 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220
phosphor-ERK(Abcam), AKT (Cell Signaling, Danvers, MA, USA), phosphorylated-AKT(Cell Signaling), p38 (Cell Signaling), phosphorylated-p38 (Cell Signaling) or monoclonal antibodies against β-actin (AC-15, Sigma Aldrich). Horseradish peroxidase-conjugated secondary antibodies were used, and protein-antibody complexes were visualized by enhanced chemiluminescence system (Amersham) [15]. The signals were quantified with ImageJ software (rsbweb.nih.gov/ij/ ).
4.6 Statistical analysis
All data were expressed as mean ± standard deviation (SD). The one- way ANOVA test was used to determine differences between groups. Any p value less than 0.05 is considered statistically significant.
5. Conclusions
In conclusion, our work has identified Lycogen™ as a anti-melanogenic agent with the capacity to ameliorate α-MSH induced-hyperpigmentation. However, we also elucidated the underlying mechanism of the therapeutic effects of Lycogen™ therapy in anti-melanogenesis.
Acknowledgments
This work was supported by grants from National Science Council (NSC 101-2320-B-039-012-MY3). Conceived and designed the experiments: WSL YDK. Performed the experiments: WSL YDK KHC. Analyzed the data: CHL. Contributed reagents/materials/analysis tools: WSL FHC CHL CHL. Wrote the manuscript: CHL
Conflict of Interest
W.S. Liu, F. H Chang and C.H. Liu are employed by Asia-Pacific Biotech Developing, Inc. (Kaohsiung, Taiwan) which produces Lycogen™. All other authors have no conflict of interest.
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