Hypopigmentary effects of 4-n-butylresorcinol and resveratrol in combination.

In the present study, the effects of 4-n-butylresorcinol and/or resveratrol on melanogenesis were studied. To achieve synergistic effects and avoid potential adverse effects, combinations of the agents in low concentrations were investigated. Our results show that 1 microM of 4-n-butylresorcinol and 1 microM of resveratrol did not individually inhibit melanin synthesis. However, the combination of 4-n-butylresorcinol (1 microM) and resveratrol (1 microM) significantly reduced melanin synthesis. Furthermore, 4-n-butylresorcinol (10 microM) and resveratrol (10 microM) decreased melanogenesis much stronger. 4-n-Butylresorcinol is reported to directly inhibit tyrosinase, the rate-limiting melanogenic enzyme, without changing tyrosinase levels. Our results also showed that resveratrol did not directly inhibit tyrosinase at 0.1-10 microM. Literature has reported that resveratrol led to post-transcriptional regulation of tyrosinase. However, Western blot analysis showed that neither 4-n-butylresorcinol nor resveratrol alone decreased tyrosinase protein levels. Surprisingly, the combination of 4-n-butylresorcinol and resveratrol reduced tyrosinase levels. Therefore, these results indicate that the synergistic hypopigmentary effect of 4-n-butylresorcinol and resveratrol results from a decreased level of tyrosinase possibly resulting from synergistic action of 4-n-butylresorcinol on tyrosinase alteration by resveratrol.

Indole-3-carbinol and ultraviolet B induce apoptosis of human melanoma cells via down-regulation of MITF.

We investigated the mechanism of indole-3-carbinol (13C)/ultraviolet B (UVB)-induced apoptosis using SK-MEL-2 and SK-MEL-5 human melanoma cells. 13C/UVB significantly reduced the viability of SK-MEL-2 cells, whereas it had little influence on SK-MEL-5 cells. Correspondingly, cell cycle analysis showed that 13C/UVB induced a clear increase in the sub-G0/G1 phase in SK-MEL-2 cells. Furthermore, 13C/UVB activated caspase-9, caspase-8, caspase-3, and Bid and caused the cleavage of poly(ADP-ribose) polymerase (PARP) in SK-MEL-2 cells. In contrast, 13C/UVB showed no effects on the apoptotic signaling pathways in SK-MEL-5 cells. Moreover, we found that 13C down-regulated the microphthalmia-associated transcription factor (MITF) in SK-MEL-2 cells, but not in SK-MEL-5 cells. Next, to investigate the involvement of MITF in 13C/UVB-induced apoptosis, MITF silencing was conducted using small interfering RNA (siRNA) for MITF in SK-MEL-5 cells. Interestingly, 13C/UVB dramatically decreased the viability of MITF-down-regulated SK-MEL-5 cells. These results indicate that MITF plays a critical role in melanoma cell survival.

Hypopigmentary action of dihydropyranocoumarin D2, a decursin derivative, as a MITF-degrading agent.

In this study, the decursin derivative dihydropyranocoumarin D2 (1) was selected for its effects on melanogenesis using a spontaneously immortalized mouse melanocyte cell line (Mel-Ab). The results showed that 1 effectively inhibited melanin synthesis in a concentration-dependent manner, but that it did not inhibit tyrosinase in a cell-free system. In addition, the changes in ERK, Akt, and microphthalmia-associated transcription factor (MITF) in response to treatment with 1 were assessed. The results revealed that ERK was dramatically up-regulated and MITF was down-regulated in response to treatment with 1, but that Akt was unchanged. Therefore, the effects of 1 on melanogenesis were examined in the absence or presence of PD98059 (a specific inhibitor of the ERK pathway). PD98059 restored hypopigmentation and the down-regulation of MITF induced by 1. Finally, MITF down-regulation by 1 was clearly restored by both chloroquine, a lysosomal proteolysis inhibitor, and MG132, a proteasome inhibitor.

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

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 mum 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.

Differential expression of p63 isoforms in normal skin and hyperproliferative conditions.

BACKGROUND: The p63 is regarded as a potential stem cell marker. METHODS: Expression of p63 isoforms was examined in normal skin and hyperproliferative conditions including psoriasis and artificial skin equivalents (SEs). Rapidly adhering (RA) and slowly adhering (SA) cells were isolated, and Western blotting was performed. RESULTS: Expression of p63 (4A4) and p63 (H-137) is similar in all conditions, although there is some variation in psoriasis. However, expression of p63alpha (C-12) is markedly different. In normal skin, p63alpha (C-12)-positive cells were scattered in whole epidermis. But in psoriasis, p63alpha (C-12)-positive cells were observed at the tips of rete ridges. In SEs, p63alpha (C-12)-positive cells were not well observed. Western blot results showed that the RA cells express p63 (4A4) and p63 (H-137) strongly compared with SA or nonadhering (NA) cells. In contrast, SA or NA cells strongly express p63alpha (C-12). CONCLUSIONS: Results suggest that both p63 (4A4) and p63 (H-137) can detect epidermal stem cells. But, p63 (H-137) seemed to be a better marker because p63 (H-137)-positive cells were more localized at basal layer. In addition, it can be said that p63alpha (C-12) can detect TAp63, which is important in differentiation of epidermis. Furthermore, it is concluded that molecular control of TAp63 is especially disorganized in hyperproliferative condition including psoriasis and SEs.

Terrein inhibits keratinocyte proliferation via ERK inactivation and G2/M cell cycle arrest.

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 p27(KIP1) (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.

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.

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.

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.

Differential regulation of melanosomal proteins after hinokitiol treatment.

BACKGROUND: Melanogenesis is regulated by a series of enzymes under the control of microphthalmia-associated transcription factor (MITF). OBJECTIVE: The aim of this study was to examine melanosome-associated protein levels in Mel-Ab cells after hinokitiol treatment. METHODS: We measured melanin contents and analyzed melanosome-associated protein levels using Western blot and RT-PCR analysis. RESULTS: Hinokitiol markedly inhibited melanin synthesis and also reduced the protein levels of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP-1), tyrosinase-related protein 2 (TYRP-2) and MITF in Mel-Ab cells. In addition, hinokitiol significantly increased the phosphorylations of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Furthermore, reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that TYR and MITF mRNA levels were significantly decreased but that levels of TYRP-1 and TYRP-2 mRNA were unaffected by hinokitiol treatment. These results suggest that hinokitiol-induced ERK phosphorylation reduces MITF and TYR transcription, and mediates the action of hinokitiol on melanogenesis. Interestingly, the mRNAs of TYRP-1 and TYRP-2 were unaffected, although the protein levels of TYRP-1 and TYRP-2 were down-regulated. Thus, the effects of hinokitiol on the transcription of TYR may differ from its effects on TYRP-1 and TYRP-2. CONCLUSION: Therefore, we suggest that TYRP-1 and TYRP-2 may be regulated by post-translational degradation after hinokitiol treatment.

Heat treatment decreases melanin synthesis via protein phosphatase 2A inactivation.

In the present study, we investigated the effects of heat treatment on melanogenesis in a mouse melanocyte cell line (Mel-Ab). It has been reported that activated extracellular signal-regulated kinase (ERK) is responsible for microphthalmia-associated transcription factor (MITF) degradation, which leads to a reduction in tyrosinase protein production and melanin synthesis. Here we demonstrate that heat treatment induces sustained ERK activation, which may inhibit melanogenesis. However, the specific ERK pathway inhibitors, PD98059 or U0126 did not restore heat-induced hypopigmentation. Furthermore, PD98059 or U0126 hardly blocked the heat-induced activation of ERK. These results suggest that heat treatment may inactivate protein phosphatase, and thus ERK activation is maintained. To support this hypothesis, we examined the effects of heat treatment on protein phosphatase 2A (PP2A) activity. The results obtained show that heat treatment inactivates PP2A, which may subsequently cause ERK activation and that heat treatment inhibits MITF promoter activity. Overall, our results demonstrate that heat treatment reduces melanin production in a temperature-dependent manner.

The fixation of living skin equivalents.

Histologic evaluations and immunohistochemical characterizations are important in studies of artificial organs such as skin equivalents. However, tissue compact organization is not easy to obtain when the artificial organ is constructed in vitro. Thus, appropriate fixation methods must be selected according to the compactness of the artificial organ and tissue engineering methodologies. The effects of several fixatives-Carnoy, Bouin's solution, formalin, paraformaldehyde, and paraformaldehyde-glutaraldehyde-were examined to select the best fixation method for preserving the structural and molecular markers of skin equivalents. Formalin-based fixatives ware found to be relatively free of the histologic problems (e.g., tissue shrinkage, poor structural preservation, weak stainability, and nonspecific immunolocalization) presented by the soft tissue fixatives (i.e., Carnoy or Bouin's solution). Unfortunately, the standard concentration of formalin induced detachment of epidermis from dermis, but this was prevented by reducing the concentration of the fixative. These findings suggest that fixation procedures should be selected for particular tissue and specific goals; in particular, they show that the paraformaldehyde-glutaraldehyde combination performed best in terms of preserving the histologic features of skin equivalents.

Cytoprotective effect of green tea extract and quercetin against hydrogen peroxide-induced oxidative stress.

In this study, we evaluated the cytoprotective effects of antioxidative substances in hydrogen peroxide (H2O2) treated Mel-Ab melanocytes. Tested substances include selenium, quercetin, green tea (GT) extract, and several vitamins (ascorbic acid, Trolox, and folic acid). Of these, both quercetin and GT extract were found to have strong cytoprotective effects on H2O2-induced cell death. We also examined additive effects, but no combination of two of any of the above substances was found to act synergistically against oxidative damage in Mel-Ab cells. Nevertheless, a multi-combination of GT extract, quercetin, and folic acid appeared to prevent cellular damage in a synergistic manner, which suggests that combinations of antioxidants may be of importance, and that co-treatment with antioxidants offers a possible means of treating vitiligo, which is known to be related to melanocyte oxidative stress.

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 irradiation via 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.

Impaired repair ability of hsp70.1 KO mouse after UVB irradiation.

UV light is absorbed in the epidermis and induces sunburn cell formation. It has been reported that HSP70 increases the UVB resistance of cell lines by in vitro experiments using various cell lines. In this study, hsp70.1(-/-) KO mouse was used in order to study the role of HSP70 after UVB irradiation. Western blotting showed a decreased level of HSP70 in hsp70.1(-/-) KO mouse compared with wild type FVB mouse. Six h after UVB irradiation, there were no significant histologic differences between the hsp70.1(-/-) KO mouse and the wild type FVB mouse. A similar degree of nuclear swelling was observed. However, there were significant differences at 12 and 24 h after UVB irradiation. After 12 h, a few apoptotic cells were observed in the wild type mouse, but a large number of cells were apoptotic in the hsp70.1(-/-) KO mouse. After 24 h, the epidermis of the wild type FVB mouse was relatively intact, but almost the entire epidermis was necrotic in the hsp70.1(-/-) KO mouse. These results showed that epidermal injury of hsp70.1(-/-) KO mouse was much more severe than that of wild type mouse although initial changes are similar in both species of mice. These results suggest that susceptibility of hsp70.1(-/-) KO mouse to UVB irradiation may originate from a defect in the repair mechanism. This HSP deficient model may be useful in studies of the effects of tissue injury that relate to the impaired tissue repair mechanisms.

Effects of lysophosphatidic acid on melanogenesis.

In this study, we investigated the effects of lysophosphatidic acid (LPA) on melanogenesis in Mel-Ab cells. We found that LPA significantly attenuates melanin synthesis, and reduces the activity of tyrosinase, the rate-limiting melanogenic enzyme. Interestingly, LPA was also found to induce the activation of a 90 kDa ribosomal S6 kinase (RSK-1), which is known to phosphorylate microphthalmia-associated transcription factor (MITF) at serine 409. Though it has been previously reported that the phosphorylation of MITF is followed by the degradation of MITF, we found that LPA significantly inhibited MITF promoter activity, and that this reduced MITF and tyrosinase protein production. Our results indicate that LPA contributes to reduced melanin synthesis via the down-regulation of MITF.

Involvement of ERK AND p38 MAP kinase in AAPH-induced COX-2 expression in HaCaT cells.

Cyclooxygenase-2 (COX-2) appears to play an important role in inflammation and carcinogenesis, and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) is a hydrophilic azo compound known to generate free radicals. Because reactive oxygen species (ROS) are known to elevate COX-2 expression, we evaluated the effect of AAPH on the expression of COX-2 in a human keratinocyte cell line, HaCaT. When cells were exposed to AAPH, marked COX-2 induction was observed. To clarify the signaling mechanism involved, we next investigated the effects of AAPH upon three major subfamilies of the mitogen-activated protein kinases (MAPKs). AAPH caused an increase in the phosphorylation of extracellular signal-regulated kinase (ERK), p38 and c-Jun NH(2)-terminal kinase (JNK). Furthermore, we found that PD98059, an ERK pathway inhibitor, and SB203580, a p38 MAPK inhibitor, diminished AAPH-induced COX-2 expression and PGE(2) production, whereas JNK inhibitor did not suppress COX-2 expression or PGE(2) production by AAPH. These findings suggest that the ERK and p38 MAPK pathways, but not the JNK pathway, are involved in AAPH-induced inflammatory progression. In addition, we found that both the water-soluble Vitamin E derivative, Trolox, and the green tea constituent, (-)-epigallocatechin gallate (EGCG), diminished AAPH-induced COX-2 expression and p38 activation.

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 degrees C) produce less melanin than cells at 37 degrees 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 degrees C for 1 h or 3 h did not affect tyrosinase activity or melanin synthesis, whereas long exposures to 31 degrees 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.

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.

(-)-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 (beta-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.