Kyoung-Chan Park

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.

Delayed ERK activation by ceramide reduces melanin synthesis in human melanocytes

Sphingolipid metabolites regulate many aspects of cell growth and differentiation. However, the effects of sphingolipids on the growth and melanogenesis of human melanocytes are not known. In the present study, we investigated the effects of sphingolipid metabolites and the possible signalling pathways involved in human melanocytes. Our data show that C(2)-ceramide inhibits cell growth in a dose-dependent manner, whereas sphingosine-1-phosphate (SPP) has no effect. Moreover, we observed that the melanin content of the cells was significantly decreased by C(2)-ceramide. The pigmentation-inhibiting effect of C(2)-ceramide at 1-10 microM was stronger than that of kojic acid, tested at 1-100 microM. The tyrosinase activity of cell extracts was reduced by C(2)-ceramide treatment. However, in the cell-free system, C(2)-ceramide could not suppress tyrosinase, whereas kojic acid directly inhibited tyrosinase. These results suggest that C(2)-ceramide decreases the pigmentation of melanocytes indirectly regulating tyrosinase. Furthermore, we found that C(2)-ceramide decreased the protein expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression. To identify the signalling pathway of ceramide, we studied the ability of C(2)-ceramide to influence extracellular signal-regulated protein kinase (ERK) and Akt/protein kinase B (PKB) activation. C(2)-ceramide induced a delayed activation of ERK ( > 1 h) and a much later activation of Akt/PKB ( > 3 h) in human melanocytes. In addition, the specific inhibition of the ERK and the Akt signalling pathways by PD98059 and LY294002, respectively, increased melanin synthesis. Thus, it seems that sustained ERK and Akt activation may lead to the suppression of cell growth and melanogenesis.

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.

A randomized controlled trial of the efficacy and safety of a fixed triple combination (fluocinolone acetonide 0·01%, hydroquinone 4%, tretinoin 0·05%) compared with hydroquinone 4% cream in Asian patients with moderate to severe melasma

Background  Melasma is an acquired, chronic hypermelanosis for which therapy remains a challenge.

Oxidation of indole-3-acetic acid by horseradish peroxidase induces apoptosis in G361 human melanoma cells

The combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) has recently been proposed as a novel cancer therapy. However, the mechanism underlying the cytotoxic effect involved is substantially unknown. Here, we show that IAA/HRP treatment induces apoptosis in G361 human melanoma cells, whereas IAA or HRP alone have no effect. It is known that IAA produces free radicals when oxidized by HRP. Because oxidative stress could induce apoptosis, we measured the production of free radicals at varying concentrations of IAA and HRP. Our results show that IAA/HRP produces free radicals in a dose-dependent manner, which are suppressed by ascorbic acid or (-)-epigallocatechin gallate (EGCG). Furthermore, antioxidants prevent IAA/HRP-induced apoptosis, indicating that the IAA/HRP-produced free radicals play an important role in the apoptotic process. In addition, IAA/HRP was observed to activate p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK), which are almost completely blocked by antioxidants. We further investigated the IAA/HRP-mediated apoptotic pathways, and found that IAA/HRP activates caspase-8 and caspase-9, leading to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. These events were also blocked by antioxidants, such as ascorbic acid or EGCG. Thus, we propose that IAA/HRP-induced free radicals lead to the apoptosis of human melanoma cells via both death receptor-mediated and mitochondrial apoptotic pathways.

Rapid healing and reduced erythema after ablative fractional carbon dioxide laser resurfacing combined with the application of autologous platelet-rich plasma.

BACKGROUND Fractional carbon dioxide laser resurfacing (FxCR) has shown considerable efficacy in reducing wrinkles, although complications such as scarring and prolonged erythema are more common and down‐time is longer than with nonablative laser treatment. Platelet‐rich plasma (PRP), a high concentration of platelets in a small volume of plasma, is known to enhance tissue healing. OBJECTIVE To evaluate the benefits of PRP in the wound healing process after FxCR. MATERIALS AND METHODS Twenty‐five subjects were treated with FxCR on the bilateral inner arms. PRP was prepared from 10 mL of whole blood and applied on a randomly allocated side, with normal saline being used as the contralateral control. Transepidermal water loss (TEWL) and skin color were measured on both sides. Skin biopsies were also taken from five subjects on day 28. RESULTS Significantly faster recovery of TEWL was seen on the PRP‐treated side. The erythema index and melanin index on the PRP‐treated side were lower than on the control side. Biopsy specimens from the PRP‐treated side showed thicker collagen bundles than those from the control side. CONCLUSION Application of autologous PRP is an effective method for enhancing wound healing and reducing transient adverse effects after FxCR treatment. The authors have indicated no significant interest with commercial supporters.

(-)-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.

Does facial sebum excretion really affect the development of acne

Background  It is generally accepted that the severity of acne is correlated with facial sebum secretion. However, previous studies on the relation between seborrhoea and the development of acne did not consider topographical differences in facial sebum secretion and used relatively vague acne severity grading systems.

Sphingosine-1-phosphate inhibits human keratinocyte proliferation via Akt/protein kinase B inactivation

Although sphingosine-1-phosphate (S1P) is a well-known mitogen, it has only recently been demonstrated that S1P is able to inhibit cell proliferation in human epidermal keratinocytes and hepatic myofibroblasts. In the present study, we investigated the possible signalling pathways involved in the growth inhibition of human keratinocytes. Our results show that S1P potently inhibits keratinocyte proliferation, and that this leads to the inhibition of DNA synthesis. Interestingly, the prolonged activation of extracellular signal-regulated protein kinase (ERK) and the transient inactivation of Akt/protein kinase B (PKB) were also observed in concert with the inhibition of keratinocyte proliferation by S1P. To verify further the antiproliferative action of S1P, we examined changes in cell cycle-related proteins. S1P inhibited cyclin D(2) synthesis but stimulated p21(WAF1/CIP1) (p21) and p27(KIP1) (p27) synthesis; all are inhibitors of cyclin-dependent kinase. Furthermore, we found that the growth inhibition by S1P was in part abolished by pertussis toxin (PTX) treatment, but that ERK activation and Akt/PKB inhibition were not abrogated, suggesting that S1P functions both intracellularly, as a second messenger, and extracellularly, as a ligand for cell surface receptors. Insulin-like growth factor I (IGF-I) is a well-established human keratinocyte mitogen and is known to stimulate Akt/PKB in various cell types. In the present study, S1P was found to inhibit the keratinocyte proliferation and Akt/PKB activation induced by IGF-I. Our results suggest that S1P may play an important role in the negative regulation of keratinocyte proliferation by inhibiting the Akt/PKB pathway.

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.