Sun-Bang Kwon

Sphingosine-1-phosphate decreases melanin synthesis via sustained ERK activation and subsequent MITF degradation

Sphingosine-1-phosphate (S1P) has emerged as a bioactive lipid modulator that mediates a variety of cell functions. However, the effects of S1P on melanogenesis are not well known. Therefore, we investigated the actions of S1P on melanin synthesis using a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. This study shows that S1P significantly inhibits melanin synthesis in a concentration-dependent manner, and also that the activity of tyrosinase was reduced in S1P-treated cells. In contrast, a specific extracellular signal-regulated protein kinase (ERK) pathway inhibitor, PD98059, increased tyrosinase activity and melanin production, and PD98059 also restored the S1P-induced reduction of tyrosinase activity and pigmentation. In addition, we found that S1P induces the sustained activation of ERK and the subsequent degradation of microphthalmia-associated transcription factor (MITF), which plays a key role in melanogenesis. Thus, we further studied the relationship between the ERK pathway and melanin synthesis. PD98059 was found to prevent the S1P-induced MITF phosphorylation and degradation and to abrogate the S1P-induced downregulation of tyrosinase and of tyrosinase-related protein 1 (TRP1) production. These results indicate that the ERK pathway is potently involved in the melanogenic signaling cascade, and that S1P-induced ERK activation contributes to reduced melanin synthesis via MITF degradation. Therefore, we suggest that S1P reduces melanin synthesis by ERK activation, MITF phosphorylation and degradation, and by the subsequent downregulation of tyrosinase and TRP-1 production.

Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents

Abstract.The isolation of human epidermal stem cells is critical for their clinical applications. In the present study, we isolated three populations of epidermal keratinocytes according to their ability to adhere to collagen type IV: i.e., rapidly adhering (RA), slowly adhering (SA), and non-adhering (NA) cells. The aim of this study was to characterize RA cells and to investigate the possibility of using these cells for epidermis reconstruction. To identify RA cells, flow cytometric analysis was performed using anti-α6 integrin and anti-CD71 antibodies. RA cells express high levels of α6 integrin and low levels of CD71, which are considered as markers of an epidermal stem cell nature. Furthermore, electron microscopy showed that RA cells are small and have a high nuclear to cytoplasmic ratio, whereas SA and NA cells have well-developed cellular organelles and abundant tonofilaments. Western blot analysis showed that RA cells are slow cycling and express p63, a putative epidermal stem cell marker, whereas SA and NA cells express c-Myc, which is known to regulate stem cell fate. To compare epidermal regenerative abilities, skin equivalents (SEs) were made using RA, SA, and NA cells. The epidermis constructed from RA cells was well formed compared to those formed from SA or NA cells. In addition, only SEs with RA cells expressed α6 integrin and β1 integrin at the basal layer. These results indicate that RA cells represent epidermal stem cells and are predominately comprised of stem cells. Therefore, the isolation of RA cells using a simple technique offers a potential route to their clinical application, because they are easily isolated and provide a high yield of epidermal stem cells.

(-)-Epigallocatechin-3-gallate and hinokitiol reduce melanin synthesis via decreased MITF production

In this study, the effects of (−)-epigallocatechin-3-gallate (EGCG) and/or hinokitiol (β-thujaplicin) on melanogenesis were investigated. Our results showed that both EGCG and hinokitiol significantly inhibited melanin synthesis in a concentration-dependent manner, and that their hypopigmenting effects were stronger than that of kojic acid, which is known to inhibit melanin formation in melanocytes and melanoma cells. Interestingly, EGCG did not show any additive hypopigmenting effect in combination with kojic acid, though EGCG did show a synergistic effect in combination with hinokitiol. Several reports indicate that the activation of extracellular signal-regulated kinase (ERK) induces microphthalmia-associated transcription factor (MITF) degradation. Accordingly, the effects of EGCG and hinokitiol on the ERK signaling pathway were examined. EGCG and hinokitiol induced neither ERK activation nor MITF degradation. On the other hand, both EGCG and hinokitiol reduced the protein levels of MITF and of tyrosinase, the rate limiting melanogenic enzyme, whereas kojic acid had no effect. In addition, hinokitiol strongly downregulated the activity of tyrosinase, whereas EGCG or kojic acid had only a little effect. These results show that both EGCG and hinokitiol reduce MITF production, and suggest that reduced tyrosinase activity by hinokitiol explains their synergistic effect on melanogenesis.

Dietary supplementation of (+)-catechin protects against UVB-induced skin damage by modulating antioxidant enzyme activities

Purpose: The aim of this study was to investigate the effects of dietary supplementation with (+)‐catechin on cutaneous antioxidant enzymes and the skin damage caused by UVB irradiation.

Hydrogen peroxide is a mediator of indole-3-acetic acid/horseradish peroxidase-induced apoptosis.

Recently, we reported that a combination of indole‐3‐acetic acid (IAA) and horseradish peroxidase (HRP) induces apoptosis in G361 human melanoma cells. However, the apoptotic mechanism involved has been poorly studied. It is known that when IAA is oxidized by HRP, free radicals are produced, and since oxidative stress can induce apoptosis, we investigated whether reactive oxygen species (ROS) are involved in IAA/HRP‐induced apoptosis. Our results show that IAA/HRP‐induced free radical production is inhibited by catalase, but not by superoxide dismutase or sodium formate. Furthermore, catalase was found to prevent IAA/HRP‐induced apoptotic cell death, indicating that IAA/HRP‐produced hydrogen peroxide (H2O2) may be involved in the apoptotic process. Moreover, the antiapoptotic effect of catalase is potentiated by NADPH, which is known to protect catalase. On further investigating the IAA/HRP‐mediated apoptotic pathway, we found that the IAA/HRP reaction leads to caspase‐3 activation and poly(ADP‐ribose) polymerase (PARP) cleavage, which was also blocked by catalase. Additionally, we found that IAA/HRP produces H2O2 and induces peroxiredoxin (Prx) sulfonylation. Consequently, our results suggest that H2O2 plays a major role in IAA/HRP‐induced apoptosis.

Long-term suppression of tyrosinase by terrein via tyrosinase degradation and its decreased expression

Abstract:  Previously, we reported that a fungal metabolite, terrein, decreases melanin synthesis via downregulation of microphthalmia‐associated transcription factor (MITF). In the present study, we further investigated the long‐term hypopigmenting action of terrein in a spontaneously immortalized mouse melanocyte cell line, Mel‐Ab. Treatment with terrein at a concentration of 50 μm strongly decreased melanogenesis in a time‐dependent manner. Interestingly, the decreased tyrosinase protein levels lasted for at least 7 days, even though the MITF protein levels were restored after 3 days of treatment. In accordance with the results of Western blot analyses, the tyrosinase mRNA levels were found to be continuously decreased for at least 7 days, even though recovery of the MITF mRNA levels began after 3 days of terrein treatment. Therefore, we evaluated tyrosinase downregulation to determine if it is caused by proteasomal degradation. We found that the reduction in tyrosinase levels that was induced by terrein was clearly recovered by MG‐132, a proteasome inhibitor. Moreover, ubiquitination of tyrosinase increased following treatment with terrein in the presence of MG‐132. Taken together, these results suggest that terrein decreases melanogenesis through ubiquitin‐dependent proteasomal degradation as well as via decreased expression of its mRNA.

Protective effects of EGCG on UVB-induced damage in living skin equivalents.

In this study, we evaluate the effects of (−)-epigallocatechin-3-gallate (EGCG) on ultraviolet B (UVB)-irradiated living skin equivalents (LSEs). Histologically, UVB irradiation induced thinning of the LSE epidermis, whereas EGCG treatment led to thickening of the epidermis. Moreover, EGCG treatment protected LSEs against damage and breakdown caused by UVB exposure. Immunohistochemically, UVB-exposed LSEs expressed p53, Fas, and 8-hydroxy-deoxyguanosine (8-OHdG), all of which are associated with apoptosis. However, EGCG treatment reduced the levels of UVB-induced apoptotic markers in the LSEs. In order to determine the signaling pathways induced by UVB, Western blot analysis was performed for both c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which are associated with UVB-induced oxidative stress. UVB activated JNK in the epidermis and dermis of the LSEs, and EGCG treatment reduced the UVB-induced phosphorylation of JNK. In addition, p38 MAPK was also found to have increased in the UVB-exposed LSEs. Also, EGCG reduced levels of the phosphorylation of UVB-induced p38 MAPK. In conclusion, pretreatment with EGCG protects against UVB irradiationvia the suppression of JNK and p38 MAPK activation. Our results suggest that EGCG may be useful in the prevention of UVB-induced human skin damage, and LSEs may constitute a potential substitute for animal and human studies.

Sphingosine-1-phosphate-induced ERK activation protects human melanocytes from UVB-induced apoptosis.

Ultraviolet B (UVB) is known to induce apoptosis in human melanocytes. Here we show the cytoprotective effect of sphingosine-1-phosphate (S1P) against UVB-induced apoptosis. We also show that UVB-induced apoptosis of melanocytes is mediated by caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, and that S1P prevents apoptosis by inhibiting this apoptotic pathway. We further investigated three major mitogen-activated protein (MAP) kinases after UVB irradiation. UVB gradually activated c-Jun N-terminal kinase (JNK) and p38 MAP kinase, while extracellular signal-regulated protein kinase (ERK) was inactivated transiently. Blocking of the p38 MAP kinase pathway using SB203580 promoted cell survival and inhibited the activation of caspase-3 and PARP cleavage. These results suggest that p38 MAP kinase activation may play an important role in the UVB-induced apoptosis of human melanocytes. To explain this cytoprotective effect, we next examined whether S1P could inhibit UVB-induced JNK and p38 MAP kinase activation. However, S1P was not found to have any influence on UVB-induced JNK or p38 MAP kinase activation. In contrast, S1P clearly stimulated the phosphorylation of ERK, and the specific inhibition of the ERK pathway using PD98059 abolished the cytoprotective effect of S1P. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that S1P may show its cytoprotective effect through ERK activation in human melanocytes.

Sphingosylphosphorylcholine inhibits melanin synthesis via pertussis toxin-sensitive MITF degradation

Objectives Sphingolipids act as structural components in cell membranes, and form lipid intermediates that have functional roles as signalling molecules in various cellular processes. Our previous findings have suggested that sphingolipid metabolites are deeply involved in the regulation of melanogenic processes. In this study we aimed to examine sphingosylphosphorylcholine‐mediated signalling pathways related to melanogenesis.

Terrein inhibits keratinocyte proliferation via ERK inactivation and G2/Mcell cycle arrest

Abstract:  Terrein, a fungal metabolite, has been recently shown to have a strong antiproliferative effect on skin equivalents. In the present study, we further investigated the effects of terrein on the possible signalling pathways involved in the growth inhibition of human epidermal keratinocytes by examining the regulations of extracellular signal‐regulated protein kinase (ERK) and of the Akt pathway by terrein. It was observed that ERK was inactivated by terrein and that keratinocyte proliferation was inhibited, whereas Akt was unaffected. The inhibition of the ERK pathway by U0126 (a specific ERK inhibitor) also had a dose‐dependent antiproliferative effect on human keratinocytes. These results indicate that ERK inhibition is involved in keratinocyte growth inhibition by terrein. Moreover, flow cytometric analysis showed that terrein inhibits DNA synthesis, as evidenced by a reduction in the S phase and an increase in the G2/M phase of the cell cycle. Thus, we next examined changes in the expressions of G2/M cell cycle‐related proteins. Terrein was found to downregulate cyclin B1 and Cdc2 without Cdc2 phosphorylation, but upregulated p27KIP1 (p27), a known inhibitor of cyclin‐dependent kinase. These results suggest that terrein reduces human keratinocyte proliferation by inhibiting ERK and by decreasing the expressions of cyclin B1 and Cdc2 complex.