Part II. The inhibition mechanism of TCTE on melanin synthesis
8. Effects of TCTE on ERK activation in melan-a melanocytes
7. Effects of TCTE on Mitf expression in melan-a melanocytes
To investigate whether TCTE does regulate Mitf expression, melan-a melanocytes were treated with 15 µM and 30 µM TCTE for 72 hrs. Mitf levels were measured by western blot. There was marked reduction of Mitf level by TCTE treatment (Figure 18).
8. Effects of TCTE on ERK activation in melan-a melanocytes
A strong activation of ERK1 and ERK2 (ERK1/2) was induced by TCTE addition in melan-a melanocytes at 60 minutes after treatment. This activation was inhibited by PD98059, a selective inhibitor of MEK (MAPK/ERK kinase) that is specific upstream activator of ERK (Figure 19).
Figure 17. Effects of TCTE on cAMP accumulation. Melan-a melanocytes were cultured to subconfluence, then incubated for 40 min in the presence of α-MSH (at 10 nM ), forskolin (For. at 20 µM) and/or TCTE (at 15 or 30 µM), then assayed for cAMP concentration as detailed in Materials and Methods; results are reported as pmol/106 cells + SD (n=4)
Figure 18. Western blot analysis of Mitf. Melan-a melanocytes were seeded into 60 mm2 culture dishes and treated with 15 and 30 µM TCTE or vehicle (0, DMSO) for 72 hrs. The cells were removed from the dishes and cell extracts electrophoresed on NuPAGET M gels and the proteins electroblotted onto nitrocellulose membrane and detected by chemiluminescence as described in Materials and Methods. Equal protein loading was checked using actin antibody.
TCTE 0 15 30 µM
Mitf
actin
Figure 19. Effects of TCTE on the activation of ERK1/2 by TCTE treatment.
Melan-a melanocytes were treated with DMSO, 10 µM PD98059 with/without 30 µM TCTE for 1 hr. Western blot was analyzed to quantify and determine the phosphorylation state of ERK1/2. Equal protein loading was checked using ERK1/2 antibody.
phospho-ERK1 phospho-ERK2
ERK1 ERK2 TCTE 30 µM - + + -
PD98059 10 µM - + - +
IV. DISCUSSION
Treatment of various pigmentary disorders and cosmetic applications require the use of depigmenting agents. Currently available topical agents used for the reduction of pigmentation includ ing tyrosinase inhibitors and melanocyte-cytotoxic agents were reviewed in Jimbow and Jimbow.87 Although substantial advances have been made, there is still an obvious need for more effective, and less irritating depigmenting therapies.
To find effective and safe depigmenting agent, seven gallic acid derivatives and two cinnamic acid derivatives were synthesized and their inhibitory activities on melanin synthesis were measured in melan-a melanocytes. Gallic acid showed depigmentation effects in me lan-a melanocytes, but it also had cytotoxic effects (Table 3). Most known depigmenting agents with small molecular weights are phenolic compounds and usually they show high cytotoxicity to the melanocytes. 87
Phenols such as hydroquinone and monobenzyl ether of hydroquinone have been reported to induce irreversible depigmentation of the skin in humans and animals.94 4-Isopropylcatechol is a potent depigmenting agent. It can be oxidized by any oxidase, including tyrosinase. Therefore, it is not a stable compound. It is highly melanocytotoxic, and this cytotoxicity derives from the interaction with toxic semiquinone/quinone radicals.87
It is possible that the oxidation of gallic acid by tyrosinase produces toxic radicals.
To reduce the cytotoxic effects of gallic acid, the hydroxyl groups were substituted with alkoxy group. The cytotoxic effect of gallic acid was reduced with the substitution of hydroxy group with alkoxy groups. In addition, the depigmenting effects were increased and cytotoxicity was reduced in proportion with the chain length of alkoxy group (Table 3). Among them TCTE was most effective in lowering melanogenesis and have a low cytotoxicity. TCTE inhibited melanin synthesis in dose-dependent manner without affecting cell proliferation. Inhib itory effect of TCTE on melanin synthesis was not only manifested in melan-a melanocytes cell but in reconstituted human epidermis and UVB- induced hyperpigmentation in brownish guinea pigs.
Although TCTE inhibited tyrosinase activity more than 70% in melan-a melanocytes with 72 h treatment (figure 12 A), this compound dose not appear to act directly on either melanosomes or tyrosinase but, rather, requires an intact cell to exert its inhibitory effect because it did not reduced tyrosinase activity in a cell free system (Figure 13). It was possible that reduced tyrosinase activity by TCTE treatment was due to down regulation of gene expression because TCTE did not inhibit tyrosinase directly but decreased tyrosinase activity in situ. To know the effects of TC TE on tyrosinase or other melanogenic enzymes like a 1 and TRP-2 expression, western blot analysis was performed on to these proteins. From the western blot analysis it was clear that the reduced tyrosinase activity was due to lowered tyrosinase protein level (figure 14). In order to examine whether TCTE affects tyrosinase and TRP-1 expression at the transcriptional levels, RT-PCR
analysis was done. From the RT-PCR analysis, it was also clear that tyrosinase was controlled by TCTE at a transcriptional level (figure 15). Tyrosinase and TRP-1 mRNA level was reduced by TCTE treatment. However, TRP-2 was not changed by TCTE treatment. Further study was done on the factors that could control the expression level of tyrosinase and TRP-1 gene expression.
cAMP has long been implicated as a key regulator of pigmentation. In murine melanoma cells, α-MSH, forskolin, IBMX, or dibutyryl cAMP which raises the intracellular concentration of cAMP was shown to induce pigmentation and increases in tyrosinase mRNA, protein and activity.56-59 Figure 16 shows that tyrosinase activity was increased by IBMX, forskolin or dbcAMP in melan-a melanocytes but the increased tyrosinase activity was down regulated to lower than control level by TCTE treatment. This result suggested that TCTE could regulate cAMP level in melan-a melanocytes.
To investigate the effects of TCTE on the cAMP level in melan-a melanocytes, cAMP level was measured after 40 minute treatment of TCTE. cAMP level was elevated 3- to 7- fold with α-MSH or forskolin treatment in melan-a melanocytes, but no change was observed with TCTE co-treatment (Figure 17). No change of cAMP level by TCTE suggested inhibitory effects of this compound on melanin synthesis may result from the down stream signal of cAMP.
Recently, the molecular events linking cAMP to melanogenesis up-regulation have been elucidated. This cascade involves the activation of protein kinase A and cAMP
responsive element binding protein (CREB) transcription factor, leading to the up-regulation of Mitf. 95 Mitf is a basic helix- loop-helix transcription factor containing a leucine- zipper domain.70 Studies on cell-specific expression of tyrosinase had shown that the M box (AGTCATGTGCT) is required for its efficient expression in melanocytes.29-30, 96 Mitf is known to trans-activate the mouse tyrosinase and trp-1 promoters, by binding to the M box, 96 well conserved in the tyrosinase gene family.
Mitf has also been known to trans-activate the human TRP-1 gene promoter, not the TRP-2 promoter through the M-box but.
Western blot analysis showed that Mitf was down regulated by TCTE treatment for 72 h in melan-a melanocytes. Therefore, the reduced expression of tyrosinase and TRP-1 can be explained by lowered Mitf level in melan-a melanocytes treated with TCTE.
It is of interest that though expression of tyrosinase and TRP-1 was down regulated by TCTE, that of TRP-2 was not changed at all. It has been reported that tyrosinase and TRP-1 are co-regulated by melanotrophic agent, which controls cAMP dependent protein kinase and protein kinase C in B16 melanoma cells.97 TRP-2 was reported to be regulated differently from tyrosinase and TRP-1, 98, 99 There is also an report that TRP-2 expression has no relations with melanin production in 10 kinds of human melanoma cell lines.100 The different response of tyrosinase, TRP-1 and TRP-2 to TCTE can be a proof that TRP-2 is regulated differently by Mitf.
From the RT-PCR and western blot analysis, it was found that though mitf mRNA expression level was not changed by TCTE treatment (Figure 15) the protein level
was decreased significantly (Figure 18). This result suggested that the decreased mitf protein level due to TCTE could result from the accelerated degradation of Mitf.
Recent studies demonstrated that phosphorylation of Mitf at serine 73 is responsible for Mitf ubiquitination and degradation.71 Melanoma cells treated with a specific inhibitor of the MAPK pathway induced melanogenesis by increase in tyrosinase expression.77 Similarly, constitutive overexpression of mutants of Ras and MEK inhibits tyrosinase expression and melanogenesis.77 These findings suggest that sustained ERK activation can suppress melanogenesis via increased Mitf degradation, which is induced by ERK-dependent Mitf phosphorylation.96 In addition, Kim et al.101 reported that delayed ERK activation by ceramide reduced melanin synthesis in human melanocytes. To investigate the effects of TCTE on ERK activation, western blot analysis was carried out. ERK phosphorylation was induced strongly at 30 min after TCTE treatment. Moreover, cotreatment with PD98059 completely blocked TCTE action on ERK activation. This result suggested that TCTE could activate ERK phosphorylation and eventually reduce melanin synthesis by accelerating Mitf degradation.
In summary, the results of this study suggested that TCTE, a synthetic ester of cinnamic acid and thymol, inhibited melanin synthesis by reducing the expression of tyrosinase and TRP-1 at the transcriptional level. The decreased expression of tyrosinase and TRP-1 resulted from the reduced Mitf level in melan-a melanocytes.
Furthermore, it is possible that reduced level of Mitf was associated with ERK1/2 activation by TCTE which accelerated degradation of Mitf in turn.
V. CONCLUSION
To find effective and safe depigmenting agents, seven gallic acid derivatives and two cinnamic acid derivatives were synthesized and their inhibitory activities on melanin production were measured using melan-a melanocytes. Among them, TCTE was most effective in inhibiting melanogenesis at concentration of 15 to 30 µM and showed little cytotoxicity at these concentrations. The inhibitory effect of TCTE on melanin synthesis was not only exerted in melan-a melanocytes but also demonstrated in human epidermal equivalent model (Melanoderm) and in brown guinea pigs with UVB- induced hyperpigmentation model. TCTE inhibited melanin synthesis by reducing the expression of tyrosinase and TRP-1 at the transcriptional level. The reduced level of Mitf might cause the down regulation of tyrosinase and TRP-1 expression in melan-a melanocytes. Furthermore, it is possible that the reduced level of Mitf was associated with ERK1/2 activation by TCTE.
From these results, it can be suggested that TCTE can be a candidate for depigmenting agent working at the upstream level of Mitf expression.
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