Seo-Hyoung Park

(-)-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.

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.

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.

Temperature regulates melanin synthesis in melanocytes

Temperature change is one of the major environmental factors that influence the human skin. However, the relationship between temperature and melanogenesis has received little attention. In the present study, we investigated the effects of temperature change on melanogenesis in a mouse melanocyte cell line (Mel-Ab), and primary cultured human melanocytes. We found that Mel-Ab cells cultured at low temperatures (31 and 34°C) produce less melanin than cells at 37°C. These results were confirmed by experiments upon human melanocytes, demonstrating that the hypopigmenting effect of low temperatures is not cell type dependent. The observed melanin production was found to be accompanied by tyrosinase activity at each temperature, indicating that tyrosinase activity is regulated by temperature. We further examined whether the incubation period at low temperatures plays an important role in the regulation of melanogenesis. Short exposures to 27°C for 1 h or 3 h did not affect tyrosinase activity or melanin synthesis, whereas long exposures to 31°C for 2 days or 6 days significantly reduced tyrosinase activity and melanin synthesis in a duration-dependent manner. Our results suggest that exposure to low temperature and the duration of this exposure are important regulators of melanogenesis.

Additive effects of heat and p38 mapk inhibitor treatment on melanin synthesis

It has been reported that the activation of extracellular signal-regulated kinase (ERK) reduces melanin synthesis. Recently, we also found that heat treatment induces ERK activation and inhibits melanogenesis in Mel-Ab cells (a mouse melanocyte cell line). In addition, it was reported that p38 MAPK (mitogen-activated protein kinase) inhibition blocks melanogenesis. Thus, we investigated the effects of heat and of the p38 MAPK inhibitor, SB203580, on melanogenesis. In this study, we found that heat treatment activates ERK and reduces melanin production in human melanocytes, and that this is accompanied by a reduction in tyrosinase activity. To regulate the ERK and p38 MAPK pathways simultaneously, we combined heat treatment and SB203580 and measured melanin synthesis. The results obtained showed that heat treatment and SB203580 reduced melanin synthesis more effectively than heat or SB203580 alone. We conclude that ERK activation and p38 MAPK inhibition can work in an additive manner to decrease melanogenesis.

The hypopigmentary action of KI-063 (a new tyrosinase inhibitor) combined with terrein

Resorcinol derivatives are known to inhibit melanin synthesis. In this study, resorcinol derivatives were synthesized and screened for their activity on melanogenesis. KI‐063 (a tyrosinase inhibitor) was examined for its effects on melanogenesis using a spontaneously immortalized mouse melanocyte cell line (Mel‐Ab). In a cell‐free system, KI‐063 directly inhibited tyrosinase, the rate‐limiting melanogenic enzyme. Moreover, in a cell system, it inhibited melanin synthesis in a concentration‐dependent manner. In addition, KI‐063 inhibited the activity of cellular tyrosinase. Thus, this study examined the effects of a combination of KI‐063 with terrein, an agent that down‐regulates microphthalmia‐associated transcription factor. The data suggest that KI‐063 has an additive effect in combination with terrein. Thus, the suppression of tyrosinase activity by KI‐063 and the inhibition of tyrosinase production by terrein appear to be an optimal combination for skin whitening.

Lysophosphatidic acid inhibits melanocyte proliferation via cell cycle arrest.

Lysophosphatidic acid (LPA) is a well-known mitogen in various cell types. However, we found that LPA inhibits melanocyte proliferation. Thus, we further investigated the possible signaling pathways involved in melanocyte growth inhibition. We first examined the regulation of the three major subfamilies of mitogen-activated protein (MAP) kinases and of the Akt pathway by LPA. The activations of extracellular signal-regulated protein kinase (ERK) and c-JunN-termi-nal kinase (JNK) were observed in concert with the inhibition of melanocyte proliferation by LPA, whereas p38 MAP kinase and Akt were not influenced by LPA. However, the specific inhibition of the ERK or JNK pathways by PD98059 or D-JNKI1, respectively, did not restore the antiproliferative effect. We next examined changes in the expression of cell cycle related proteins. LPA decreased cyclin D1 and cyclin D2 levels but increased p21WAF1/CIP1 (p21) and p27KIP1 (p27) levels, which are known inhibitors of cyclin-dependent kinase. Flow cytometric analysis showed the inhibition of DNA synthesis by a reduction in the S phase and an increase in the G0/G1 phase of the cell cycle. Our results suggest that LPA induces cell cycle arrest by regulating the expressions of cell cycle related proteins.