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Pigmentation in the dermis is known to be caused by melanophages, defined as melanosome-laden macrophages. In this study, we show that dermal fibroblasts also have an ability to uptake melanosomes and apoptotic melanocytes. We have previously demonstrated that normal human melanocytes constantly secrete melanosome clusters from various sites of their dendrites. After adding secreted melanosome clusters collected from the culture medium of melanocytes, time-lapse imaging showed that fibroblasts actively attached to the secreted melanosome clusters and incorporated them. Annexin V staining revealed that phosphatidylserine (PtdSer), which is known as an 'eat-me' signal that triggers the internalization of apoptotic cells by macrophages, is exposed on the surface of secreted melanosome clusters. Dermal fibroblasts were able to uptake secreted melanosome clusters as did macrophages, and those fibroblasts express TIM4, a receptor for PtdSer-mediated endocytosis. Further, co-cultures of fibroblasts and melanocytes demonstrated that dermal fibroblasts internalize PtdSer-exposed apoptotic melanocytes. These results suggest that not only macrophages, but also dermal fibroblasts contribute to the collection of potentially toxic substances in the dermis, such as secreted melanosome clusters and apoptotic melanocytes, that have been occasionally observed to drop down into the dermis from the epidermis.

Placental extracts have been widely used as skin lightening agents in the Japanese cosmetic market. Here, we show that placental extracts contain factors that can decrease or increase melanin synthesis by normal human melanocytes in vitro in possible association with mitochondrial respiration. When normal human melanocytes were treated with a whole porcine placental extract, melanin synthesis was decreased. In contrast, a porcine placental extract in which exudates and insoluble materials including lipids had been removed increased melanin synthesis. In addition, the amount of tyrosinase, the enzyme critical for melanin synthesis, changed in accordance with the alteration of melanin synthesis. Interestingly, the amount of manganese-dependent superoxide dismutase (MnSOD), a mitochondrial-resident antioxidant enzyme, was increased when melanin synthesis was decreased by the whole placental extract. Mitochondrial respiration and glycolysis also changed following treatment with the placental extracts. These results suggest that placental extracts contain factors that can increase or decrease melanin synthesis by normal human melanocytes and that mitochondrial function may be associated with the placental extract-induced regulation of melanogenesis., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

Recent studies have described the role of shedding vesicles as physiological conveyers of intracellular components between neighboring cells. Here we report that melanosomes are one example of shedding vesicle cargo, but are processed by a previously unreported mechanism. Pigment globules were observed to be connected to the filopodia of melanocyte dendrites, which have previously been shown to be conduits for melanosomes. Pigment globules containing multiple melanosomes were released from various areas of the dendrites of normal human melanocytes derived from darkly pigmented skin. The globules were then captured by the microvilli of normal human keratinocytes, also derived from darkly pigmented skin, which incorporated them in a protease-activated receptor-2 (PAR-2)-dependent manner. After the pigment globules were ingested by the keratinocytes, the membrane that surrounded each melanosome cluster was gradually degraded, and the individual melanosomes then spread into the cytosol and were distributed primarily in the perinuclear area of each keratinocyte. These results suggest a melanosome transfer pathway wherein melanosomes are transferred from melanocytes to keratinocytes via the shedding vesicle system. This packaging system generates pigment globules containing multiple melanosomes in a unique manner.

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 (IC(50)=200μM) was significantly less effective at inhibiting tyrosinase from NHEM. DP decreased melanin content in cultured NHEM after 7th day (IC(50)=10μM). The IC(50) 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 © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

One of the major activities of melanocytes in skin is to produce melanin and transport it via dendrites to neighboring keratinocytes. Here, we present evidence that Rab8, a member of the small GTPase superfamily, is present in purified melanosomal fractions, and is upregulated by pigmentogenic agents like melanocyte-stimulating hormone/isobutylmethyl xanthine (MSH/IBMX) and ultraviolet radiation B (UVB). Confocal immunofluorescence microscopic studies revealed that Rab8 is colocalized with Mel5, a melanosomal protein, at the trans-Golgi area and in the cytoplasmic vesicles of B16 cells. During MSH/IBMX treatment, while a number of dendrites with numerous processes are formed, colocalization is extended towards the tips of protrusions. Since process formation is supported by cytoskeletal assembly as well as membrane transport, we tested the colocalization of Rab8 with actin filaments in B16 cells. Rab8, indeed, colocalized with phalloidin, mostly at the periphery, but when irradiated with UVB, cells were rounded instead of dendritic, and colocalization was found predominantly at the cytoplasmic area. Further, suppression of Rab8 expression by its antisense oligonucleotide revealed the reduction in staining intensity of Rab8 but not of Mel5, dendrite formation and melanosome transport towards the tips of the dendrites in B16 melanoma cells. Taken together, it is suggestive that Rab8, in B16 melanoma cells, might have a role in melanosome traffic and dendrite extension, both in constitutive and regulated fashion.

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]

Presents a study which examined the effect of α-Tocopheryl ferulate on the pigmentation of normal human melanocytes. Materials and methods; Results; Discussion.

Demonstrates the association of small guanosine triphosphate-binding protein Rab3A with melanosomes in pigment cells. Isolation of melanosome enriched fraction; Pathways of transferring melanin pigment outside of the cytosol of melanocytes; Information on intracellular vesicle trafficking.

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.

Summary: Background: Vitamin C, which is a strong anti-oxidant, plays an important role in maintaining physiological states. In dermatology, Vitamin C is used for treatment of various skin problems such as de-pigmentation of hyperpigmented spots. However, Vitamin C has limited stability and permeability, and development of a Vitamin C derivative with improved properties is needed. Objective: We evaluated the effect of a lipophilic Vitamin C derivative, tetra-isopalmitoyl ascorbic acid (VC-IP), on ultraviolet (UV)-induced skin pigmentation, to determine its potential as a more effective form of Vitamin C. Methods: The release of Vitamin C from VC-IP was examined using a reconstructed skin model following topical application of VC-IP. Anti-oxidative and anti-inflammatory activities of VC-IP were tested in cultured human keratinocytes. Subsequently, clinical test was done to clarify the effect of VC-IP on UVB-induced skin pigmentation. Results: VC-IP released Vitamin C in physiological conditions and worked as pro-Vitamin C. In subsequent experiments, we found that VC-IP suppressed the elevation of intracellular peroxide after UVB irradiation, and enhanced cellular tolerance against UVB and reactive oxygen species such as hydrogen peroxide and tert-butyl hydroperoxide. Furthermore, VC-IP reduced the production of interleukin-1α and prostaglandin E2 in UVB-irradiated keratinocytes and suppressed melanocyte proliferation in conditioned culture medium prepared from UVB-irradiated keratinocytes. Finally, in a clinical study, topical application of a 3% VC-IP cream for 3 weeks suppressed pigmentation after UVB irradiation. Conclusions: These results demonstrate that VC-IP is a precursor of Vitamin C, and effectively suppresses UVB-induced skin pigmentation, possibly through its anti-oxidative activity. [Copyright &y& Elsevier]

Normal human melanocytes are often grown in vitro in the continuous presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) for growth in vitro. The expression of protein kinase C (PKC) subspecies, which are the major cellular receptors for phorbol esters, was examined in melanocytes after long-term treatment with TPA to investigate the role of PKC subspecies in TPA-dependent cell growth. The PKC enzyme activity detected in quiescent melanocytes was almost completely depleted in cells after incubation with 85 nM TPA for 48 h. Immunoblot analysis indicated that, among the PKC subspecies α, β, δ, ε, and ζ expressed in quiescent cells, α-, β-, δ-, and ε-PKC were significantly down-regulated, whereas ζ-PKC remained at detectable levels in TPA-treated cells. TPA did not significantly affect the expression or subcellular distribution of ζ-PKC in melanocytes. Immunoprecipitation assay revealed that the enzyme activity of ζ-PKC was increased in both the cytosol and particulate cell fractions, but the increase was much greater in the latter. The activation of ζ-PKC lasted for 24 to 48 h after the addition of TPA; thereafter, ζ-PKC activity returned to basal levels. DNA synthesis was shown to change concomitantly with the activation of ζ-PKC in TPA-treated cells. These results indicate that TPA induces not only the down-regulation of α-, β-, δ-, and ε-PKC, but also long-term activation of ζ-PKC in melanocytes, and that activation of ζ-PKC parallels the growth of normal human melanocytes. [ABSTRACT FROM AUTHOR]