[6]-Shogaol Inhibits α-MSH induced Melanogenesis through the Acceleration of ERK and PI3K/Akt -Mediated MITF Degradation
Huey-Chun Huang1, Shu-Jen Chang2, Chia-Yin Wu3, Hui-Ju Ke3,Tsong-Min Chang3*
1 Department of Medical Laboratory Science and Biotechnology, College of Health Care, China Medical University, Taichung, Taiwan.
2School of Pharmacy, China Medical University, Taichung, Taiwan.
3Department of Applied Cosmetology, HungKuang University, Taichung, Taiwan.
* Corresponding author: Dr. Tsong-Min Chang
Address: No. 1018, Sec. 6, Taiwan Boulevard, Taichung, Taiwan (R.O.C.) 43302. Tel: +886-4-26318652 Ext. 2216/5309. Fax: +886-4-26315843
E-mail: [email protected]
Short title: [6]-Shogaol suppresses melanogenesis Abstract
[6]-Shogaol is the main biologically active component of ginger. The study was 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
aimed to evaluate the potential skin whitening mechanisms of [6]-shogaol. The effects of [6]-shogaol on cell viability, melanin content, tyrosinase activity, as well as the expression of the tyrosinase and microphthalmia-associated transcription factor (MITF) were measured. The results revealed that [6]-shogaol effectively suppresses tyrosinase activity and the amount of melanin and that those effects are more pronounces than those of arbutin. It was also found that [6]-shogaol decreased the protein expression levels of tyrosinase-related protein 1 (TRP-1) and microphthalmia-associated transcriptional factor (MITF). In addition, the MITF mRNA levels were also effectively decreased in the presence of 20 μM [6]-shogaol. The degradation of MITF protein was inhibited by the MEK-inhibitor (U0126) orphosphatidylinositol-3-kinase inhibitor (PI 3K inhibitor) (LY294002). Further immunofluorescence staining assay implied the involvement of the proteasome in the downregulation of MITF by [6]-shogaol. Our confocal assay results also confirmed that [6]-shogaol inhibited α-melanocyte stimulating hormone (α-MSH) induced melanogenesis through the acceleration of extracellular responsive kinase (ERK) and phosphatidylinositol-3-kinase (PI3K/Akt)-mediated MITF degradation.
Key words: [6]-Shogaol, tyrosinase, melanin, MITF, ERK, PI3K 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Introduction
Edible phytochemicals are now considered to be safe, inexpensive, readily
acceptable and promising depigmentation agents. Ginger rhizome (Zingiber officinale Roscoe) is one of the oldest herbs that is officially listed in the traditional Chinese Pharmacopoeia. [6]-Shogaol, the major shogaol in ginger rhizomes , has been found to possess many interesting pharmacological and physiological activities, such as anti-proliferation and antioxidant activities .As a traditional “antioxidant” agent, the anti-melanogenesis activity of [6]-shogaol has not been well documented. Previous studies have supported the potential of [6]-shogaol as a melanogenesis inhibitor agent. In this study, we explored the possible mechanisms of [6]-shogaol on melanin synthesis.
The biosynthesis of melanin is a complicated process involving many factors. Alpha-melanocyte stimulating hormone (α-MSH), the most important melanocyte-stimulating hormone in melanocyte-stimulating melanogenesis, promotes melanin synthesis. α-MSH binds to melanocortin 1 receptor (MC1R), which induces the activation of adenylyl cyclase, followed by cAMP production . cAMP leads to phosphorylation of CREB transcription factors, which in turn stimulate microphthalmia transcription factor (MITF) promoter activation, which is the most critical transcription factor that regulates melanocyte function. MITF directly binds to the promoter regions of melanin production genes and positively regulates their transcription; these genes 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57
include at least three melanocyte-specific enzymes: tyrosinase, tyrosinase-related protein 1 (TRP1) and tyrosinase-related protein 2 (TRP2) . Tyrosinase catalyzes the production of melanin and other pigments from tyrosine by oxidation . TRPs may be important in the regulation of melanogenesis or in the assembly of the melanogenic apparatus . Because MITF is considered a master regulator of melanogenesis,
understanding the aforementioned signaling pathways in modulating MITF function is important. α-MSH induces MITF expression by activating several signaling pathways. Activation of the classical mitogen-activated protein kinase (MAPK) pathway also plays a key role in the regulation of MITF activation and the cAMP pathway . The activation of ERK phosphorylates MITF, which is followed by MITF ubiquitination and degradation [9]. In addition, p38 MAPK has recently been shown to be involved in UVR-induced melanogenesis and leads to the activation of MITF expression and consequently increased tyrosinase expression . Furthermore, the MITF gene is transcriptionally upregulated by phosphatidylinositol-3-kinase (PI3K) signaling in melanocytes and is thus tightly regulated in a signal-dependent fashion; this in turn affects MITF expression and the regulation of pigmentation. In addition to
transcriptional regulation, MITF is also subject to various post-translational
modifications, particularly phosphorylation and degradation by ERK , ribosomal S6 kinase (RSK), glycogen synthase kinase-3b (GSK3b) and p38 .
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It has been reported that some antioxidants, such as kojic acid and ascorbic acid derivatives , play an important role in the inhibition of melanogenesis, which implies the close association between antioxidative mechanisms and the down-regulation of hyperpigmentation. Recently, the anti-melanogenic effects of [6]- and [8]-gingerol have been reported . However, the effects of other components of ginger rhizome on the melanogenesis signaling pathway have not been investigated. In this study, we examined the effect of [6]-shogaol on α-MSH-induced melanogenesis in mouse melanoma B16F10 cells. In particular, we analyzed changes in the ERK signaling pathway and the associated MITF regulation.
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Materials and Methods Materials
[6]-shogaol (Fig. 1) and kojic acid were purchased from Wako pure chemical Industries (Osaka, Japan). All other chemicals and solvents were obtained from Sigma-Aldrich (Saint Louis, MO).
Cell culture
B16F10 cells (ATCC CRL-6475; BCRC60031) were cultured in Dulbecco's
modification of Eagle's medium (DMEM) with 10% fetal bovine serum (FBS; Gibco, Langley, OK, USA) and penicillin/streptomycin (100 I.U/50 μg/mL) (Sigma
Chemical Co, Saint Louis, MO, USA) in a humidified atmosphere containing 5% CO2 in air at 37℃.
Trypan blue exclusion assay
A trypan blue exclusion assay was used to assess the effect of [6]-shogaol on
B16F10 cell growth and viability. Briefly, following treatment with [6]-shogaol, cells were trypsinized and pelleted by centrifugation, and the cell pellet was re-suspended in 300 μL of DMEM media. Trypan blue (0.4% in PBS, 10 μL) was added to a smaller aliquot (20 μL) of cell suspension, and the cell number (viable unstained and nonviable blue) was counted using a hemocytometer under the microscope. Each sample was counted in triplicate, and each experiment was repeated at least three times. 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105
Tyrosinase activity assay
The B16F10 cellular tyrosinase activity was determined according to a previously described method . The cells were treated with α-MSH (100 nM) for 24 h, and then further treated with various concentrations of [6]-shogaol (1, 5, 10 and 20 mg/mL) or arbutin (2.0 mM) for another 24 h. After these treatments, the cells were washed twice with PBS and homogenized with 50 mM PBS (pH 7.5) buffer containing 1.0 % Triton X-100 and 0.1 mM PMSF (phenylmethylsulfonyl fluoride; a serine proteinase
inhibitor). Intracellular tyrosinase activity was monitored as follows. The cellular extracts (100 μL) were mixed with freshly prepared L-DOPA solution (0.1% in phosphate-buffered saline) and incubated at 37°C for 30 min. The absorbance at 490 nm was measured with a Gen 5TM microplate reader (BIO-TEK Instrument,
Winooski, VT) to monitor the production of dopachrome. Melanin content measurement
The B16F10 cells were first stimulated with α-MSH (100 nM) for 24 h and then further treated with chemical inhibitors (10 μM) or combined with [6]-shogaol for an additional 24 h. After the treatments, the cells were detached by incubation in
trypsin/EDTA and subsequently centrifuged at 5,000 g for 5 min. The cell pellets were then solubilized in 1 N NaOH at 60°C for 60 min. The melanin content was assayed by spectrophotometric analysis at 405 nm absorbance.
Western blot analysis 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125
Cells were lysed in PBS containing 1% Nonidet P-40, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 5 μg/mL aprotinin, 100 μg/mL
phenylmethylsulfonyl fluoride, 1 μg/mL pepstatin A, and 1 mM
ethylenediaminetetraacetic acid (EDTA) at 4°C for 20 min. Total lysates were quantified using a microBCA kit (Thermo Fisher Scientific, Rockford, IL). Proteins (20 μg) were resolved by SDS-polyacrylamide gel electrophoresis and
electrophoretically transferred to a PVDF membrane. The membrane was blocked in 5% fat-free milk in PBST buffer (PBS with 0.05 % Tween-20) followed by incubation overnight with the following primary antibodies diluted in PBST buffer: rabbit anti-mouse MITF antibody (1:1000), rabbit anti-anti-mouse TRP1 antibody (1:2000), rabbit anti-mouse TRP2 antibody (1:1000), rabbit anti-mouse AKT antibody (1:1000), rabbit anti-mouse p-AKT antibody (1:1000), rabbit anti-mouse β-actin antibody (1:10,000), rabbit anti-mouse ERK antibody (1:1000), rabbit anti-mouse p-ERK antibody
(1:1000) (Santa Cruz Biotech, Dallas, TX), and rabbit anti-mouse tyrosinase antibody (1:3000), (Epitomics, Burlingame, CA). The primary antibodies were removed, and the membrane was washed extensively in PBST buffer. Subsequent incubation with horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:20000, Santa Cruz Biotech, Dallas, TX) was performed at room temperature for 2 h. The membrane was washed extensively in PBST buffer to remove any excess secondary antibodies, and 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144
the blot was visualized with Enhanced ChemiLuminescence reagent (GE Healthcare Piscataway, NJ).
Two-step RT-PCR
RNA samples were reverse-transcribed for 120 min at 37°C with High Capacity cDNA Reverse Transcription Kit according to the standard protocol detailed by the supplier (Applied Biosystems). Quantitative PCR was performed as follows: 10 min at 95°C, and 40 cycles of 15 sec each at 95°C, 1 min at 60°C using 2X Power SYBR Green PCR Master Mix (Applied Biosystems) and 200 nM of forward and reverse primers. Each assay was run on an Applied Biosystems 7300 Real-Time PCR system in triplicate and expression fold-changes were determined using the comparative CT method. Relative quantification was performed using GAPDH as an endogenous control.
Immunofluorescence staining
B16F10 cells were fixed with 4% paraformaldehyde in 250 mM Hepes, pH 7.4, freshly diluted from 16% stocks stored at –20°C. After 5 min at room temperature, the cells were washed with phosphate-buffered saline and treated for 1 h at 4°C with blocking solution (PBST, 1% FBS). Gels were incubated with primary antibodies in blocking solution overnight at 4°C in a wet chamber. After washing in PBST, the cells were incubated with appropriate secondary antibody mixtures in blocking solution for at least 1 h at room temperature. The cells were washed three times in 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164
PBS and mounted in Gold antifade reagent with DAPI (Life Technologies, Carlsbad, CA). Confocal analysis was performed using a Leica TCS SP2 confocal microscope: 10 horizontal scans using a 63× (1.3 NA) oil immersion objective were recorded for each image with the imaging software (exported as a TIFF file).
Statistical analysis
The statistical significance of differences was evaluated by ANOVA test after examining the variances; p < 0.05 (marked as “*”), p<0.01 (marked as “**”), or p<0.001(marked as “***”) was considered to be statistically significant.
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Results
The cells treated with various concentrations of [6]-shogaol (1, 5, 10 or 20 μM) for 24 h or 48 h were evaluated for cell viability based on the trypan blue exclusion assay. The results are expressed as percent viability relative to control (Fig. 2). The different concentrations of [6]-shogaol exhibited non-cytotoxic effects on B16F10 cell
viability. Hence, we applied 20 μM of [6]-shogaol in the subsequent cell experiments. When B16F10 cells were cultured in medium containing [6]-shogaol, the
stimulation of tyrosinase activity by α-MSH was reduced to 87%, 68%, 58% and 43% by 1, 5, 10 and 20 μM [6]-shogaol, respectively. The IC50 of [6]-shogaol was 15.75 μM. In addition, the intracellular tyrosinase activity was 64% in the arbutin-treated cells (Table 1). Thus, [6]-shogaol acts as a potential tyrosinase inhibitor. Additionally, the melanin content induced by α-MSH was decreased to 72% by 20 μM [6]-shogaol. The residual melanin content in arbutin-treated cells was 66% of control. We also investigated the dependence of the inhibitory effect of [6]-shogaol on the ERK and PI3K signaling pathways. Cells pretreatment with α-MSH were further treated with MEK(U0126), ERK (PD98059) or PI3K (LY294002) inhibitors with[6]-shogaol (Table 1). Cells added with MEK or ERK signal inhibitors exhibited increased melanin content compared to cells treated only with [6]-shogaol. The same pattern was evident using LY294002. The results showed that [6]-shogaol signaling through 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191
ERK and PI3K inhibits B16F10 intracellular tyrosinase activity and subsequently decreases melanin production.
To elucidate the mechanism of the influence of [6]-shogaol on melanogenesis, the the expression of tyrosinase, TRP1, TRP2, and MITF in α-MSH-stimulated B16F10 cells were analyzed by western blotting. B16F10 cells were incubated with 100 nM of α-MSH and then exposed to the [6]-shogaol at the specified time points. [6]-Shogaol inhibited α-MSH-induced MITF expression to 0.68, 0.55 and 0.4-fold of control at 16 h, 24 h, and 48 h, respectively (Fig. 3A). The inhibitory effects of [6]-shogaol on MITF production are much lower than inhibitory effects of the effective level (200 μM) of kojic acid (KA), which is an established effective melanogenesis inhibitor . This inhibition was correlated with down-regulation of TRP-1 expression by 0.53-fold at 48 h. In contrast, [6]-shogaol treatment did not display changes in the tyrosinase and TRP2 levels in α-MSH-treated B16F10 cells. Decreased MITF mRNA levels were also detected in the presence of 20 μM [6]-shogaol (Fig. 3B).
To further confirm the role of ERK and PI3K signaling pathways in [6]-shogaol induced inhibition of melanogenesis, three kinase inhibitors were incubated before they were exposed to [6]-shogaol. Cells pre-treated with LY294002 or U0126 before stimulation with [6]-shogaol displayed increased expression of MITF compared to cells treated only with [6]-shogaol by increasing AKT or ERK phosphorylation, 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210
respectively (Fig. 4). The MITF expression in B16F10 cells co-treated with α-MSH and PD98059 was higher than in the cells treated with α-MSH alone. However, the synergistic effect of α-MSH and PD98059 on the MITF was decreased by the [6]-shogaol treatment. These results suggested that the [6]-[6]-shogaol-induced
anti-melanogenic effect may be mediated by activation of the AKT and ERK pathway. In addition, the protein expression levels of p-ERK and p-AKT were also increased by [6]-shogaol (Fig. 4). Furthermore, blocking p-ERK and PI3K increased the melanin content attenuated by [6]-shogaol (Table 1). These results demonstrate that the MITF inhibitory effect of [6]-shogaol is dependent on the ERK and PI3K signaling
pathways.
The down-regulation of MITF expressions by [6]-shogaol was also investigated by immunofluorescence staining assay. As shown in Fig. 5, the fluorescence signal for MITF was mainly observed in the nuclei of B16F10 cells. Immunofluorescent staining showed a substantial decrease in overall MITF staining after 24 h of incubation with [6]-shogaol; this staining was absent in the nucleus and showed a diffuse cytosolic distribution after [6]-shogaol treatment. Downregulation of MITF was prevented by pre-treatment with proteasome inhibitor MG-132, suggesting the involvement of the proteasome in the downregulation of MITF by [6]-shogaol. We also used a specific inhibitors, U0126 and PD98059, which were able to reverse the 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229
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