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Excess production of melanin or its abnormal distribution, or both, can cause irregular hyperpigmentation of the skin, leading to melasma and age spots. To date, various quasi-drugs that prevent or improve hyperpigmentary disorders have been developed and officially approved by the Ministry of Health, Labor and Welfare of Japan. Many of these inhibit the activity of tyrosinase, an enzyme required for melanin synthesis, for example, by competitive or non-competitive inhibition of its catalytic activity, by inhibiting its maturation, or by accelerating its degradation. In this review, we categorize the quasi-drugs developed in Japan to prevent or treat hyperpigmentary disorders, or both, and discuss perspectives for future development. [ABSTRACT FROM AUTHOR]

Pigmentation of the skin, hair and eyes is regulated by tyrosinase, the critical rate-limiting enzyme in melanin synthesis by melanocytes. Tyrosinase is degraded endogenously, at least in part, by the ubiquitin proteasome system (UPS). Several types of inherited hypopigmentary diseases, such as oculocutaneous albinism and Hermansky-Pudlak syndrome, involve the aberrant processing and/or trafficking of tyrosinase and its subsequent degradation which can occur due to the quality-control machinery. Studies on carbohydrate modifications have revealed that tyrosinase in the endoplasmic reticulum (ER) is proteolyzed via ER-associated protein degradation and that tyrosinase degradation can also occur following its complete maturation in the Golgi. Among intrinsic factors that regulate the UPS, fatty acids have been shown to modulate tyrosinase degradation in contrasting manners through increased or decreased amounts of ubiquitinated tyrosinase that leads to its accelerated or decelerated degradation by proteasomes. [ABSTRACT FROM AUTHOR]

Analyzes the expression of genes for tyrosinase, tyrosinase-related protein 1, tyrosinase-related protein 2 and melanocortin-1 receptor using reverse transcription polymerase chain reaction. Result of the incubation of melanocyte-stimulating hormone/isobutylmethylxanthine; Correlation of the abundance and gel mobility pattern with changes in activity in hybrids and parental melanoma cells.

The distribution of γ-glutamyl transpeptidase (γ-GTP), tyrosinase, and 5-S-cysteinyldopa (5-S-CD) within melanoma cells has been studied in vitro as well as in vivo. Sodium periodate treatment of intact B-16 melanoma cells has been found to inhibit γ-GTP present as an ectoenzyme, However, these periodate-treated cells in the presence of 10-5 M dopa and glutathione have been found to continue to secrete large quantifies of 5-S-CD in their medium. The large-granule fraction of Greene's melanotjc melanoma contains substantial amounts of both tyrosinase and γ-GTP. However, further separation of the large-granule fraction into sub-fractions indicates that tyrosinase and γ-GTP seem to co-exist with premelanosome. It is suggested that glutathione-dependent 5-S-CD genesis proceeds within premelanosomes through the formation of glutathione-dopa. The excess of glutathione-dopa and 5-S-CD, unutilized for pheomelanin formation overflow into the cytoplasm. [ABSTRACT FROM AUTHOR]

Tyrosinase, a copper-containing glycoprotein, is the rate-limiting enzyme critical for melanin biosynthesis in specialized organelles termed melanosomes that are produced only by melanocytic cells. Inhibitors of tyrosinase activity have long been sought as therapeutic means to treat cutaneous hyperpigmentary disorders. Multiple potential approaches exist that could control pigmentation via the regulation of tyrosinase activity, for example: the transcription of its messenger RNA, its maturation via glycosylation, its trafficking to melanosomes, as well as modulation of its catalytic activity and/or stability. However, relatively little attention has been paid to regulating pigmentation via the stability of tyrosinase, which depends on its processing and maturation in the endoplasmic reticulum and Golgi, its delivery to melanosomes and its degradation via the ubiquitin-proteasome pathway and/or the endosomal/lysosomal system. Recently, it has been shown that carbohydrate modification, molecular chaperone engagement, and ubiquitylation all play pivotal roles in regulating the degradation/stability of tyrosinase. While such processes affect virtually all proteins, such effects on tyrosinase have immediate and dramatic consequences on pigmentation. In this review, we classify melanogenic inhibitory factors in terms of their modulation of tyrosinase function and we summarize current understanding of how the quality control of tyrosinase processing impacts its stability and melanogenic activity.Journal of Investigative Dermatology (2007) 127, 751–761. doi:10.1038/sj.jid.5700683; published online 11 January 2007 [ABSTRACT FROM AUTHOR]

Omeprazole is a proton pump inhibitor used in the treatment of peptic ulcer disease and gastrosophageal reflux disease and acts by irreversibly blocking ATP4A, a P-type H+/K+ ATPase in gastric parietal cells. We found that omeprazole and its closely related congeners inhibited melanogenesis at micromolar concentrations in B16 mouse melanoma cells, normal human epidermal melanocytes, and in a reconstructed human skin model. Omeprazole topically applied to the skin of UV-irradiated human subjects significantly reduced pigment levels after 3 weeks compared with untreated controls. Omeprazole had no significant inhibitory effect on the activities of purified human tyrosinase or on the mRNA levels of tyrosinase, dopachrome tautomerase, Pmel17, or MITF mRNA levels. Although melanocytes do not express ATP4A, they do express ATP7A, a copper transporting P-type ATPase in the trans-Golgi network that is required for copper acquisition by tyrosinase. ATP7A relocalization from the trans-Golgi network to the plasma membrane in response to elevated copper concentrations in melanocytes was inhibited by omeprazole. Omeprazole treatment increased the proportion of EndoH sensitive tyrosinase, indicating that tyrosinase maturation was impaired. In addition, omeprazole reduced tyrosinase protein abundance in the presence of cycloheximide, suggestive of increased degradation. Our findings are consistent with the hypothesis that omeprazole reduces melanogenesis by inhibiting ATP7A and by enhancing degradation of tyrosinase. [ABSTRACT FROM AUTHOR]

Abstract: Background: 1-(2,4-Dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylpheny)propane (DP) was reported as a novel tyrosinase inhibitor by Nesterov et al. In previous study, we showed that DP is an antioxidant and accelerates the fading of UVB-induced tan in human skin but details of inhibiting mechanism of DP in melanogenesis remain incomplete. Objective: To clarify additional mechanisms of DP inhibition of melanogenesis, we studied the effect of DP on tyrosinase processing and degradation. Methods: Tyrosinase inhibition was assessed using mushroom and human tyrosinase. The effect of DP on mRNA and protein levels as well as glycosylation and degradation of tyrosinase was examined using normal human epidermal melanocytes (NHEM). Results: DP was 200 times more potent than that of kojic acid in inhibiting mushroom tyrosinase activity. In contrast, DP (IC50 =200μM) was significantly less effective at inhibiting tyrosinase from NHEM. DP decreased melanin content in cultured NHEM after 7th day (IC50 =10μM). The IC50 for DP against human tyrosinase activity was found to be at least 20 times higher than that of melanin synthesis. At a non-cytotoxic concentration DP did not decrease tyrosinase mRNA however protein level decreased by 46% after 48h treatment. DP did not alter the ratio of mature and immature tyrosinase assayed by endo H cleavage. Tyrosinase degradation assays revealed that DP accelerated tyrosinase degradation in NHEM. Conclusions: We found that DP acts through dual mechanisms to reduce melanin synthesis; by inhibition of tyrosinase activity via an anti-oxidant effect, and, more importantly, by the acceleration of tyrosinase degradation. [Copyright &y& Elsevier]

The involvement of phospholipase D (PLD) in the regulation of melanogenesis was examined. Treatment of B16 mouse melanoma cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) resulted in the activation of PLD and a decrease in melanin content. 1-Butanol, but not 2-butanol, completely blocked the TPA-induced inhibition of melanogenesis, suggesting the involvement of PLD in this event. Reverse transcription-PCR and immunoblot analyses revealed the existence of both PLD isozymes, PLD1 and PLD2, in B16 cells. When PLD1 or PLD2 was introduced into those cells by an adenoviral gene-transfer technique, both PLD1 and PLD2 were activated by TPA. When PLD1 and PLD2 were overexpressed, PLD2 potently caused a decreased in melanin content, whereas the effect of PLD1 expression on melanin content, whereas the effect of PLD1 expression on melanin content was minimal. Over-expression of PLD2 itself did not affect protein kinase C activity, as assessed by the intracellular distribution and levels of expression of each isoform expressed in B16 cells. The effects of TPA on the down-regulation of basal or αmelanocyte-stimulating hormone-enhanced melanogenesis were almost completely blocked by expressing a lipase activity-negative mutant, LN-PLD2, but not by LN-PLD1. Further, the PLD2-induced decrease in melanin content was accompanied by a decrease in the amount and activity of tyrosinase, a key enzyme in melanogenesis, whereas the mRNA level of tyrosinase was unchanged by the over-expression of PLD2. Moreover, treatment with proteasome inhibitors completely blocked the PLD2-induced down-regulation of melanogenesis. Taken together, the present results indicate that the TPA-induced down-regulation of melanogenesis is mediated by PLD2 but not by PLD1 through the ubiquitin proteasome-mediated degradation of tyrosinase. This suggests that PLD2 may play an important role in regulating pigmentation in vivo. [ABSTRACT FROM AUTHOR]

Fatty acids are common components of biological membranes that are known to play important roles in intracellular signaling. We report here a novel mechanism by which fatty acids regulate the degradation of tyrosinase, a critical enzyme associated with melanin biosynthesis in melanocytes and melanoma cells. Linoleic acid (unsaturated fatty acid, C18:2) accelerated the spontaneous degradation of tyrosinase, whereas palmitic acid (saturated fatty acid, C16:0) retarded the proteolysis. The linoleic acid-induced acceleration of tyrosinase degradation could be abrogated by inhibitors of proteasomes, the multicatalytic proteinase complexes that selectively degrade intracellular ubiquitinated proteins. Linoleic acid increased the ubiquitination of many cellular proteins, whereas palmitic acid decreased such ubiquitination, as compared with untreated controis, when a proteasome inhibitor was used to stabilize ubiquitinated proteins. Immunoprecipitation analysis also revealed that treatment with fatty acids modulated the ubiquitination of tyrosinase, i.e. linoleic acid increased the amount of ubiquitinated tyrosinase whereas, in contrast, palmitic acid decreased it. Furthermore, confocal immunomicroscopy showed that the colocalization of ubiquitin and tyrosinase was facilitated by linoleic acid and diminished by palmitic acid. Taken together, these data support the view that fatty acids regulate the ubiquitination of tyrosinase and are responsible for modulating the proteasomal degradation of tyrosinase. In broader terms, the function of the ubiquitin-proteasome pathway might be regulated physiologically, at least in part, by fatty acids within cellular membranes. [ABSTRACT FROM AUTHOR]

Coated vesicles have been found to contain much higher tyrosinase and γ-glutamyl transpeptidase activities than pre-melanosomes. This indicates that similar to tyrosinase, γ-glutamyl transpeptidase, an enzyme responsible for pheomelanogenesis, is highly concentrated in coated vesicles after its maturation in Golgi associated endoplasmic reticulum (GERL). Furthermore, in the pre- and post-dopaquinone melanogenic pathway, coated vesicles convert dopachrome to colorless indole compounds mere quickly than in premelanosomes because of their higher dopachrome conversion factor activity. Melanosomes have been found to exhibit indole conversion factor activity, while coated vesicles show indole blocking factor activity. In moderately tyrosinase-rich premelanosomes, the levels of dopachrome conversion factor and indole blocking factor are lower than in coated vesicles or melanosomes. High levels of indole blocking factor in coated vesicles may indicate why melanin polymer formation does not occur there in vivo despite their high tyrosinase activity. [ABSTRACT FROM AUTHOR]