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Analysis of the Effects of Hydroquinone and Arbutin on the Differentiation of Melanocytes
Yu InoueSeiji HasegawaTakaaki YamadaYasushi DateHiroshi MizutaniSatoru NakataKayoko MatsunagaHirohiko Akamatsu
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2013 年 36 巻 11 号 p. 1722-1730

詳細
Abstract

Hydroquinone (HQ) is a chemical compound that inhibits the functions of melanocytes and has long been known for its skin-whitening effect. According to previous studies, the Tyrosinase (Tyr) activity inhibitory effect and melanocyte-specific cell toxicity are known depigmenting mechanisms; however, details of the underlying mechanisms are unknown. Arbutin (Arb) is also known for its Tyr activity inhibitory effect and is commonly used as a skin-whitening agent. However, the detailed depigmenting mechanism of Arb is also not yet fully understood. Few studies have attempted to elucidate the effects of HQ and Arb on undifferentiated melanocytes. In this study, we examined the effects of HQ and Arb throughout each stage of differentiation of melanocytes using a mouse embryonic stem cell (ESC) culture system to induce melanocytes. The results showed that HQ in particular downregulated the early stage of differentiation, in which neural crest cells were generated, and the late stage of differentiation, in which melanogenesis became active. On the other hand, Arb had no effect on the differentiation of melanocytes, and only suppressed melanogenesis by specifically suppressing elevations in Tyr expression in the late stage of differentiation.

Melanocytes in the epidermis of the skin produce melanin, which is responsible for determining the color of human skin and hair. The generation and differentiation of melanocytes have been studied for many years and are now well understood; for example, melanocytes originate from undifferentiated neural crest cells derived from the neural tube during the embryonic stage,1) these cells then migrate to the dermis and epidermis until they mature and ultimately colonize hair follicles. Melanocyte precursors in hair follicles partially exist as undifferentiated melanocyte stem cells around the bulge area. These melanocyte stem cells have been shown to proliferate and differentiate into mature melanocytes as necessary and colonize the epidermis and hair follicles.24) In addition, mature melanocytes transfer melanosomes to peripheral keratinocytes and hair matrix cells, which may affect the color of skin and hair.5) Given these findings, melanocytes undergo diverse stages of differentiation.

If an abnormality occurs in the differentiation and proliferation of melanocytes and the mechanism of melanin synthesis, it may cause various diseases; for example, when a mutation occurs in microphthalmia-associated transcription factor (MITF-M), which is a master gene of melanocytes seen in neural crest cells immediately after their migration from the neural tube, it may cause the abnormal differentiation and proliferation of neural crest cells, resulting in Waardenburg syndrome type 2, which is characterized by white spots on the skin, iris heterochromia, and deafness.6) Paired box gene 3 (PAX3) and SRY-box containing g gene 10 (SOX10) are expressed in the neural tube before the first neural crest cells delaminate, and regulate MITF-M expression. When mutations occur in PAX3 and SOX10, it may cause Waardenburg syndrome types 1, 3, and 4.6)

Melanin arises from the amino acid, tyrosine. Tyrosinase (Tyr) catalyzes three different reactions in the biosynthetic pathway of melanin: 1) the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA); 2) the oxidation of DOPA to DOPA quinone; and 3) the oxidation of 5,6-dihydroxyindole (DHI) to indole-quinone.7) When these proteins develop abnormalities and lose their activity, melanin production does not occur, which eventually causes oculocutaneous albinism type I (OCA1).6)

The most important role of melanin is to protect the skin against UV (ultraviolet) radiation. Melanosomes are transferred from melanocytes to neighboring keratinocytes in order to form perinuclear melanin caps that protect DNA from UV damage. Melanocytes are important for the human body; however, when melanogenesis becomes hyperactive in melanocytes, the skin consequently develops epidermal hyperpigmentation including melasma, freckles, and senile lentigines. UV is one of the major external factors that affects the proliferation and differentiation of melanocytes. In mice, factors such as Endothelin 1 (Edn1), Kit ligand (Kitl), and granulocyte/macrophage colony-stimulating factor (GM-CSF) are secreted from epidermal keratinocytes and fibroblasts in response to a stimulus from ultraviolet A (UVA) and ultraviolet B (UVB), and promote the differentiation and proliferation of melanocytes.8) Senile lentigines appear as a result of an increase in melanocytes that have been matured due to UV exposure and constantly repeated melanin synthesis.

When an abnormality occurs in the generation, differentiation, and melanin synthesis of melanocytes, it can cause various types of pigmentation disorders or pigmented spots on the skin. The high visibility of these skin diseases negatively impacts on the quality of life of patients. Therefore, it is necessary to understand the mechanism controlling the differentiation and proliferation of melanocytes and to search for materials capable of controlling abnormal melanocytes.

Hydroquinone (HQ) is a chemical compound that inhibits the functions of melanocytes and has long been known for its skin-whitening effect. According to previous studies, the Tyr activity inhibitory effect and melanocyte-specific cell toxicity are known depigmenting mechanisms9,10); however, details of the underlying mechanisms are unknown. Arbutin (Arb), which is a hydroquinone glycoside, is also known for its Tyr activity inhibitory effect1113) and is commonly used as a skin-whitening agent. However, the detailed depigmenting mechanism of Arb is also not yet fully understood.

Conventional studies have used mature melanocytes in experiments to analyze the effects of HQ and Arb on melanocytes. However, to our knowledge, there was hardly any study that used undifferentiated melanocytes. Therefore, the effects of HQ and Arb on differentiation processes remain completely unknown. Not only mature melanocytes, but also melanocytes undergo various stages of differentiation (including neural crest cells, melanocyte stem cells, and melanoblasts) in the human body. We consider it of importance to analyze the effects of HQ and Arb on the differentiation process of immature melanocytes.

ES cells are pluripotent cells derived from the inner cell mass of a blastocyte, having indefinite proliferative potential and multipotency.1417) Yamane et al. established a melanocyte inducement system using mouse ES cells in 1999.18) Their inducement system has made it possible to replicate the development and differentiation processes of melanocytes and analyze the effects of materials on each development stage. We previously searched for materials that could control the differentiation of melanocytes using this melanocyte inducement system.19)

In this study, we examined the effects of HQ and Arb on the development to maturation processes of melanocytes using this melanocyte differentiation inducement system. The results showed that HQ in particular downregulated the early stage of differentiation, in which neural crest cells were generated, and the late stage of differentiation, in when melanogenesis became active. On the other hand, Arb had no effect on the differentiation of melanocytes, and only suppressed melanogenesis by specifically suppressing elevations in Tyr expression in the late stage of differentiation.

MATERIALS AND METHODS

Cell Culture

BRUCE-4 ESCs (MILLIPORE, Billerica, MA, U.S.A.), derived from mouse ESCs of the cell line C57BL/6J, were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, U.S.A.) supplemented with 15% fetal bovine serum (FBS) (Sigma, St. Louis, MO), ES Cell Qualified l-Glutamine Solution (CHEMICON International, Inc., Temecula, CA, U.S.A.), ES Cell Qualified 2-Mercaptoethanol (CHEMICON), ES Cell Qualified Non-Essential Amino Acids (CHEMICON), ES Cell Qualified Nucleosides (CHEMICON), and ESGRO (CHEMICON), according to the manufacturer’s protocols. Mouse embryonic fibroblasts (MEF) (MILLIPORE) treated with 10 µg/mL of Mitomycin C (Sigma) were used as feeder layer cells.

ST2 cells (Riken Cell Bank, Ibaraki, Japan) were maintained in Minimum Essential Medium Alpha Modification (Invitrogen) supplemented with 10% FBS.

To induce the differentiation into melanocytes, ST2 cells were used as feeder layer cells. ESCs were seeded on ST2 feeder layer cells in 24 well plates (500 cells/well), and were cultured in Minimum Essential Medium Alpha Modification supplemented with 10% FBS, 100 nM dexamethasone (Sigma), 20 pM basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, U.S.A.), 10 pM cholera toxin (Bio Academia, Osaka, Japan), and 100 ng/mL Endothelin 3 (Edn3) (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Microscopic observations revealed the formation of mouse ESC colonies on ST2 feeder layer cells on day 6 of the culture and pigmented melanocytes around these colonies on approximately day 18. Melanin synthesis was accelerated from day 18 through to day 24. We previously conducted detailed gene expression analyses using the ESC culture system and identified that the expression of each melanocyte marker was progressively increased (Supplementary Fig. 1).20)

HQ (Wako) was dissolved in ethanol at a concentration of 1 M as a stock solution, and was then added to cell cultures at a final concentration of 3–100 µM. Arb (Tokyo Chemical Industry, Tokyo, Japan) was dissolved in sterile water at a concentration of 100 mM as a stock solution, and was then added to cell cultures at a final concentration of 30–1000 µM.

Quantitative Real-Time Polymerase Chain Reaction (PCR) Analysis

During the differentiation of ESC into melanocytes, total RNA was extracted from cells at various stages using TRIZOL® Reagent (Invitrogen), and cDNA was synthesized by reverse transcription. Real-time PCR was performed with the SuperScript™ III Platinum® Two-Step qRT-PCR kit (Invitrogen), using the 7300 Real Time PCR System (Applied Biosystems, Tokyo, Japan) according to the manufacturer’s protocols. The primer sequences used were as follows:

The contents of the selected genes were normalized to Gapdh. All PCR products were checked by melting curve analysis to exclude the possibility of multiple products or an incorrect product size. PCR analyses were conducted in triplicate for each sample.

Cell Viability Assay

ESCs and ST2 cells were seeded in 96 well plates (1×104 cells/well), and the culture medium was replaced the next day by each culture medium containing HQ and Arb at various concentrations. After 24 h, relative cell numbers were measured using Cell Counting Kit-8 (DOJINDO Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. In brief, after removing the medium, 100 µL of CCK-8 solution was added to cells, and they were then incubated for another hour. Optical density values were tested at an absorbance of 450 nm using a microplate reader (Molecular Devices, Inc., Menlo Park, CA, U.S.A.).

Melanin Content Measurement

ESC and ST2 cells were co-cultured in 24 well plates and induced to differentiate into melanocytes. The melanin content per relative cell number under each culture condition was determined after 24 d of induction. In brief, after removing the medium, 300 µL of CCK-8 solution was added to the cells, and they were then incubated for another hour. A total of 100 µL of each lysate was put in another 96 well plate, and optical density values were tested at an absorbance of 450 nm by the microplate reader. After calculating the relative cell number, the melanin content of cultured cells was measured. Cells were washed twice with phosphate buffered saline (PBS), lysed in 300 µL of 2 n NaOH, and boiled for 2 h at 60°C to solubilize the melanin. A total of 200 µL of each lysate was put in another 96 well plate, and absorbance at 475 nm was measured using a microplate reader (Molecular Devices).

Statistical Analysis

The Student’s t-test was used for statistical analysis, and multiple groups were evaluated by a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison.

RESULTS

1. Effects of HQ and Arb on the Viabilities of ESCs and ST2 Cells

First, we examined the effects of HQ and Arb on the viabilities of each cell (ESC and ST2 cells). The results showed that 100 µM of HQ significantly inhibited the proliferation of ES cells and ST2 cells (Fig. 1a). On the other hand, Arb did not inhibit the proliferation of ES cells or ST2 cells to the same level as it did at 1000 µM (Fig. 1b).

Fig. 1. Effects of HQ and Arb on the Cell Proliferation of ESCs and ST2 Cells

(a, b) Relative cell numbers (%). ESCs and ST2 cells were cultured with various concentrations of HQ (a) and Arb (b). The relative cell numbers were measured in each well after 24 h. Data are expressed as the mean±S.D. of three experiments, ** p<0.01, significantly different from the control.

2. Effects of HQ and Arb on Melanin Synthesis

To analyze the effects of HQ and Arb on the differentiation of melanocytes, we co-cultured ESCs and ST2 cells in 24 well plates and induced them to differentiate into melanocytes while continuously adding HQ and Arb (HQ: 6.25, 12.5, and 25 µM, Arb: 125, 250, and 500 µM). After 24 d of induction, the melanin content per relative cell number under each culture condition was determined.

HQ and Arb showed no inhibitory effect on cell growth; however, the melanin content per relative cell number decreased in a concentration-dependent manner (Figs. 2a, b). Subsequent studies were conducted at a concentration of 25 µM of HQ and 500 µM of Arb due to their high efficacies.

Fig. 2. Effects of HQ and Arb on Melanin Synthesis

(a) Representative dishes after induction. (b) Relative cell numbers (%) and melanin content/relative cell numbers (%). ESCs were differentiated with HQ (6.25, 12.5, and 25.0 µM) and Arb (125, 250, and 500 µM). Relative cell numbers and melanin content were measured in each dish after 24 d. The figure represents relative cell numbers and melanin content/relative cell numbers normalized to relative cell numbers. Data are expressed as the mean±S.D. of three experiments, * p<0.05, ** p<0.01, significantly different from the control.

3. Analysis of the Effects of HQ and Arb, Added at Each Stage of Differentiation, on Melanin Synthesis after 24-d Differentiation Induction

In order to identify the effects of HQ and Arb at each stage of differentiation, we added HQ and Arb to the melanocyte differentiation inducement system in a specific pattern (days 0–6, days 6–12, days 12–18, and days 18–24), and analyzed their effects on terminal melanin synthesis after a 24-d culture.

The results showed that the final amount of melanin produced was significantly decreased when HQ was added on days 0–6 and days 18–24 of induction (conditions No. 1, 4–7, and 9–15, Fig. 3a). On the other hand, no downregulation in melanogenesis was observed when HQ was added on days 6–18 (conditions No. 2, 3, and 8, Fig. 3a).

Fig. 3. Effect of the Time of HQ and Arb Addition on Melanin Synthesis on Day 24

(a) Melanin content/relative cell number (%) under conditions No. 1 to No. 15. HQ (25 µM) and Arb (500 µM) were added at various times during melanocyte induction and we measured the melanin content and relative cell numbers of each dish on day 24. The figure represents the melanin content normalized to relative cell numbers. Data are expressed as the mean±S.D. of three experiments. * p<0.05, ** p<0.01, significantly different from the control.

When Arb was added on days 18–24 (conditions No. 4, 7, 9, 10, and 12–15, Fig. 3b), the final amount of melanin produced was significantly decreased. On the other hand, no downregulation in melanogenesis was observed when Arb was added on days 0–18 (conditions No. 1–3, 5, 6, 8, and 11, Fig. 3b).

HQ and Arb showed no inhibitory effect on cell growth under each culture condition (Supplementary Fig. 2).

Based on the above findings, it was suggested that HQ specifically showed suppressive effects in the early stage of differentiation, in which neural crest cells were generated (days 0–6), and the late stage of differentiation, in which melanogenesis became active (days 18–24). It was also suggested that Arb showed no effects on days 0–18, but had suppressive effects exclusively on the stage of melanogenesis (days 18–24).

4. Analysis of the Effects of HQ and Arb, Added at Each Stage of Differentiation, on the Expression of Marker Genes

In order to analyze the effects of HQ and Arb on melanocyte-specific genes, we performed detailed gene expression analyses under conditions No. 5, No. 10, and No. 15 in which the compounds were added at the early stage of induction (days 0–12), at the late stage of induction (days 12–24), and throughout the entire period (days 0–24), respectively.

The final amount of melanin produced in the HQ group was decreased under conditions No. 5, No. 10, and No. 15 (Figs. 3a, 4a). The results also showed that the increased expression of each marker was markedly downregulated under condition No. 5, while the expression of each marker was gradually increased in the control group (Fig. 4b). Downregulation in the expression of each marker was also observed under condition No. 15 (Fig. 4d). The final amount of melanin produced under condition No. 10 was significantly decreased (Fig. 3a), whereas significant changes in the expression of all marker genes were not observed (Fig. 4c).

Fig. 4. Effect of the Time of HQ Addition on the Expression Profiles of Melanocyte Cell Lineage Markers

(a) Images of representative dishes under conditions No. 5, 10, 15, and the control. (b–d) Real-time PCR for melanocyte cell lineage markers observed under conditions No. 5, 10, 15, and the control. The expression level of each marker gene was normalized to that on day 24 (terminally differentiated melanocytes of the control). Data are expressed as the mean±S.D. of three experiments. * p<0.05, ** p<0.01, significantly different from the control. Symbols: open circles, conditions No. 5, 10, and 15; closed squares, control.

The final amount of melanin produced in the Arb group was decreased under conditions No. 10 and No. 15 (Figs. 3b, 5a). The results also showed that the expression of all markers was gradually increased under condition No. 5, similar to the control (Fig. 5b). On the other hand, no significant change was observed in the expression of most marker genes, while a significant suppression was reported in the expression of Tyr under conditions No. 10 and No. 15 (Figs. 5c, d).

Fig. 5. Effect of the Time of Arb Addition on the Expression Profiles of Melanocyte Cell Lineage Markers

(a) Images of representative dishes under conditions No. 5, 10, 15, and the control. (b–d) Real-time PCR for melanocyte cell lineage markers observed under conditions No. 5, 10, 15, and the control. The expression level of each marker gene was normalized to that on day 24 (terminally differentiated melanocytes of the control). Data are expressed as the mean±S.D. of three experiments. * p<0.05, ** p<0.01, significantly different from the control. Symbols: open circles, conditions No. 5, 10, 15; closed squares, control.

Based on the above results, the expression of all melanocyte differentiation markers was significantly downregulated when HQ was added in the early stage of differentiation. On the other hand, these results demonstrate that while Arb had no effect on the differentiation of melanocytes, it specifically suppressed the increased expression of Tyr in the differentiation process.

DISCUSSION

In this study, we examined the effects of HQ and Arb on the development to maturation processes of melanocytes using a melanocyte differentiation inducement system. When HQ and Arb were added continuously to the melanocyte differentiation inducement system using ES cells, the final amount of melanin produced was decreased in a concentration-dependent manner (Figs. 2a, b). In order to identify the effects of HQ and Arb on each stage of the differentiation of melanocytes, we added HQ and Arb to the melanocyte differentiation inducement system at each stage of differentiation and induced differentiation for 24 d. We then analyzed its effects on terminal melanin synthesis.

The results showed that HQ in particular downregulated the early stage of differentiation, in which neural crest cells were generated (days 0–6), and the late stage of differentiation, in which melanogenesis became active (days 18–24) (Fig. 3a). We believe that a downregulation in the final amount of melanin produced occurred when HQ was added on days 0–6 because HQ may have inhibited the early differentiation of ES cells. A previous study reported that HQ has embryotoxicity.21) Therefore, HQ can also be considered to affect the early differentiation of ES cells and promote differentiation into other cells besides melanocyte lineage cells.22) However, very few studies have attempted to elucidate the effects of HQ on the early differentiation of ES cells.

Benzene metabolites including hydroquinone are known to affect the various gene expression levels of cells.23) Such effects of HQ may have inhibited the expression of genes necessary for melanocyte differentiation in the present study. More detailed analyses should be conducted in the future to identify why HQ controlled ES cells in the early stage of differentiation into melanocytes.

Nevertheless, we consider that ES cells lost their ability to differentiate into melanocytes because of the inhibitory effect of HQ on the early stage of differentiation of ES cells. Therefore, the expression of each melanocyte marker was not increased even after HQ was removed.

Although the final amount of melanin produced was not decreased when HQ was added on days 6–12 and days 12–18 of induction, the final amount of melanin produced was decreased when HQ was added on days 18–24 of induction when melanogenesis became active. A previous study demonstrated that HQ competitively inhibited the activation of Tyr, by acting as an alternative substrate of tyrosine.9) Therefore, we concluded that HQ became highly effective after day 18 when Tyr activity was activated. The final amount of melanin produced under condition No. 10 was significantly decreased (Fig. 3a); however, significant changes were not observed in the expression of melanocyte markers (Fig. 4c). A previous study showed that HQ did not affect gene expression related to melanin synthesis such as Tyr.24)

Arb had no effects on days 0–18; however, the final amount of melanin produced was decreased on days 18–24 only when melanogenesis became active (Fig. 3b). Since no change was observed in the expression of markers, for example, Pax3, Sox10, Mitf-M, Dct, and Tyrp1 (Figs. 5b–d), Arb was considered to have had no effects on the differentiation of melanocytes. In contrast, Arb markedly suppressed the expression of Tyr (Figs. 5c, d). Previous studies have shown that Arb works as a competitive inhibitor of Tyr and has no effect on the expression of Tyr, Tyrp1, or Dct itself.1113) These differences may have occurred because other studies used terminally-differentiated melanocytes and melanoma cells to examine the effect of Arb, while we used melanocytes that were in the process of differentiation. Arb may downregulate the expression of Tyr, in addition to the competitive inhibitory effect of Tyr against differentiating melanocytes.

Furthermore, previous studies have shown that Arb has multiple effects on cells, besides Tyr activity inhibitory effect, such as antioxidant and anti-inflammatory effects.25,26) The expression of Tyr in this study may also have been indirectly suppressed by these functions of Arb. SB203580, a p38 MAP kinase inhibitor, is known to suppress the gene expression of Tyr in melanoma cells.27) Further studies should be conducted to elucidate the mechanism by which Arb inhibited the mRNA expression of Tyr in this study.

In the present study, no significant difference was observed in the final melanin content (Figs. 3a, b) between when the expression of all melanocyte differentiation markers was decreased after HQ was continuously added (Fig. 4d) and when only the expression of Tyr was decreased after Arb was continuously added (Fig. 5d). These results may be attributed to Tyr being a key regulator of melanogenesis.7) Therefore, we consider that, even if the differentiation of melanocytes had progressed in the Arb group, melanogenesis did not occur properly because of the decreased expression of Tyr, resulting in the same final melanin content as that in the HQ group.

In this study, we analyzed the effects of HQ and Arb on the differentiation of melanocytes using the mRNA expression of each melanocyte differentiation marker as indicators. On the other hand, the protein-level effects of HQ and Arb on each gene are also of importance; therefore, we need to elucidate these details through further research in the future.

Many researchers have focused on analyzing the effects of HQ and Arb on melanogenesis. However, the effects of HQ and Arb on differentiation processes remained completely unknown. In this study, we consistently examined the effects of HQ and Arb on melanocytes from their development to maturation using a melanocyte inducement system from ES cells, and found that each agent exhibited different effects depending on the stage of differentiation. Mature and also undifferentiated melanocytes have been reported in the human body.24) Exploring specific compounds that can control undifferentiated melanocytes as well as mature melanocytes may help achieve fundamental control over skin pigmentation.

Acknowledgements

We would like to sincerely thank Ms. M. Hori (Cellisis Co., Ltd., Aichi, Japan) and Ms. M. Hotta (Fujita Health University Institute, Aichi, Japan) for their support in analyzing our experimental data.

REFERENCES
 
© 2013 The Pharmaceutical Society of Japan
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