Melanin Formation In Melanocytes Research Articles

In vitro inhibitory effect of sulfated galactans isolated from red alga Gracilaria fisheri on melanogenesis in B16F10 melanoma cells

Hyperpigmentation of the skin results from excessive melanin formation in melanocytes, and overproduction of melanin frequently leads to melanoma. The aim of this study was to investigate the potential of sulfated galactans (SG) from Gracilaria fisheri to inhibit melanin formation or melanogenesis. Cellular and molecular mechanisms of inhibition were also investigated. SG was evaluated in vitro for its inhibitory effect on mushroom tyrosinase activity, cell-free tyrosinase activity, and cellular tyrosinase activity. B16F10 mouse melanoma cells were cultured with SG and their tyrosinase activity and melanin content was compared with kojic acid, a known tyrosinase inhibitor. Moreover, the levels of expression of melanogenesis-related genes and proteins were determined by quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The result showed that SG had no inhibitory effect on mushroom tyrosinase activity and cell-free tyrosinase activity. However, SG significantly suppressed cellular tyrosinase activity and melanin production in B16F10 melanoma cells without any apparent cytotoxicity. Quantitative RT-PCR and ELISA revealed that SG downregulated the expression of microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), and tyrosinase mRNA and proteins. Taken together, the data suggest that SG may act as an anti-melanogenic agent by inhibiting the expression of MITF and cellular tyrosinase activity. SG may show potential as an ingredient in skin-whitening cosmetics or as a topical agent for the treatment of hyperpigmentation disorders.

PGC-1 Coactivators Regulate MITF and the Tanning Response

The production of pigment by melanocytes tans the skin and protects against skin cancers. UV-exposed keratinocytes secrete α-MSH, which then activates melanin formation in melanocytes by inducing the microphthalmia-associated transcription factor (MITF). We show that PPAR-γ coactivator (PGC)-1α and PGC-1β are critical components of this melanogenic system in melanocytes. α-MSH signaling strongly induces PGC-1α expression and stabilizes both PGC-1α and PGC-1β proteins. The PGC-1s in turn activate the MITF promoter, and their expression correlates strongly with that of MITF in human melanoma cell lines and biopsy specimens. Inhibition of PGC-1α and PGC-1β blocks the α-MSH-mediated induction of MITF and melanogenic genes. Conversely, overexpression of PGC-1α induces pigment formation in cell culture and transgenic animals. Finally, polymorphism studies reveal expression quantitative trait loci in the PGC-1β gene that correlate with tanning ability and protection from melanoma in humans. These data identify PGC-1 coactivators as regulators of human tanning.

Inhibition of melanogenesis by piceid isolated from Polygonum cuspidatum

Piceid (5,4'-dihydroxystilbene-3-O-β-D-glucopyranoside) is one of the stilbenes found in Polygonum cuspidatum. Previous studies have shown that this compound has little effect on tyrosinase inhibition when compared with other stilbenes in a cell-free tyrosinase assay. Furthermore, its role for melanogenesis in melanocytes is relatively unknown. In melanocytes, piceid inhibits tyrosinase activity and melanin production in a concentration-dependent manner. To explore the action of piceid on melanogenesis, we studied its effect on several key cellular enzymes and a transcriptional factor known to be involved in melanogenesis, including: tyrosinase, tyrosinase-related protein 1, tyrosinase-related protein 2, and microphthalmia-associated transcription factor. Interestingly, the effects of piceid on hypopigmentation and inhibition of tyrosinase activity were better than those of arbutin, which is well known to inhibit melanin formation in melanocytes. In addition, piceid suppressed the mRNA and protein expression of the aforementioned enzymes and transcriptional factor in a concentration-dependent manner. In this regards, our results showed that piceid represents a safe and new candidate for a skin-lightening agent.

LPS induces melanogenesis through p38 MAPK activation in human melanocytes

We have observed that lipopolysaccharide (LPS) induces pigmentation in melanocytes and in this study have examined whether these responses are mediated by the p38 mitogen-activated protein kinase (MAPK) signaling pathway. LPS appears to stimulate the pigmentation of melanocytes and cultured skin. LPS was found to induce the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase protein in cells. Stimulation of melanocytes with LPS led to time dependent phosphorylation of p38 MAPK. Furthermore, p38 MAPK functionally regulated the LPS-induced melanin formation in melanocytes; a p38 MAPK inhibitor, SB203580, almost completely attenuated the LPS-mediated up-regulation of melanin synthesis and induction of MITF and tyrosinase expression. These findings indicate that activation of p38 MAPK plays an important role in LPS-induced melanogenesis by up-regulating MITF and tyrosinase expression.

Methyl-β-cyclodextrin, a specific cholesterol-binding agent, inhibits melanogenesis in human melanocytes through activation of ERK

Cholesterol has been suggested to regulate cell differentiation. In this study, we have examined the effects of cholesterol modulation on pigmentation of skin using a treatment with methyl-beta-cyclodextrin (MbetaCD), a specific cholesterol-binding agent. Treatment with MbetaCD reduced pigmentation in human melanocyte and cultured skin. This decrease in pigmentation was related to the inhibition of the expression of tyrosinase and microphthalmia-associated transcription factor of melanocytes. Stimulation of melanocytes with MbetaCD led to the time-dependent phosphorylation of extracellular signal-regulated kinase (ERK). Furthermore, ERK functionally regulated the MbetaCD-induced melanin formation in melanocytes; a ERK inhibitor, PD98059, almost completely attenuated the MbetaCD-mediated inhibition of melanin synthesis and down-regulation of MITF and tyrosinase expression. These results suggest that cholesterol reduction by MbetaCD inhibit melanin synthesis via ERK activation and subsequent MITF downregulation.

(−)-Epigallocatechin-3-gallate and hinokitiol reduce melanin synthesisvia 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.

The inhibitory effect of glycolic acid and lactic acid on melanin synthesis in melanoma cells.

Alpha-hydroxy acids (AHAs) such as glycolic acid (GA) and lactic acid (LA) have been reported to be effective in treating pigmentary lesions such as melasma, solar lentigines, and postinflammatory hyperpigmentation. The mechanism of this effect might be due to epidermal remodeling and accelerated desquamation, which would result in quick pigment dispersion. However, the direct effect of AHAs on melanin synthesis has not yet been well studied. To elucidate such a direct effect of AHAs on melanogenesis, we performed melanin assays, growth curve determinations, Northern and Western blotting for melanogenic proteins [tyrosinase, tyrosinase related protein (TRP)-1 and TRP-2], and tyrosinase and, 4-dihydroxyphenylalaninechrome tautomerase enzyme activity assays using mouse B16 and human melanoma cells. GA or LA (at doses of 300 or 500 microg/ml) inhibited melanin formation in similar dose-dependent manner, without affecting cell growth. Although the mRNA and protein expression or molecular size of tyrosinase, TRP-1 and TRP-2 were not affected, tyrosinase activity was inhibited. To see whether GA and/or LA directly inhibit tyrosinase catalytic function, the effect of GA and LA on human tyrosinase purified from the melanosome-rich large granule fraction of human melanoma cells was performed. GA or LA were shown to inhibit tyrosinase enzyme activity directly, but this effect was not due to the acidity of GA or LA, because adjusting the pH to 5.6 (the pH of GA and LA at concentrations of 2500 microg/ml), did not affect tyrosinase activity. Taken together, these results show that GA and LA suppress melanin formation by directly inhibiting tyrosinase activity, an effect independent of their acidic nature. GA and LA might work on pigmentary lesions not only by accelerating the turnover of the epidermis but also by directly inhibiting melanin formation in melanocytes.

Glabrene and isoliquiritigenin as tyrosinase inhibitors from licorice roots.

Tyrosinase is known to be a key enzyme in melanin biosynthesis, involved in determining the color of mammalian skin and hair. Various dermatological disorders, such as melasama, age spots, and sites of actinic damage, arise from the accumulation of an excessive level of epidermal pigmentation. The inadequacy of current therapies to treat these conditions as well as high cytotoxicity and mutagenicity, poor skin penetration, and low stability of formulations led us to seek new whitening agents to meet the medical requirements for depigmenting agents. The inhibitory effect of licorice extract on tyrosinase activity was higher than that expected from the level of glabridin in the extract. This led us to test for other components that may contribute to this strong inhibitory activity. Results indicated that glabrene and isoliquiritigenin (2',4',4-trihydroxychalcone) in the licorice extract can inhibit both mono- and diphenolase tyrosinase activities. The IC(50) values for glabrene and isoliquiritigenin were 3.5 and 8.1 microM, respectively, when tyrosine was used as substrate. The effects of glabrene and isoliquiritigenin on tyrosinase activity were dose-dependent and correlated to their ability to inhibit melanin formation in melanocytes. This is the first study indicating that glabrene and isoliquiritigenin exert varying degrees of inhibition on tyrosinase-dependent melanin biosynthesis, suggesting that isoflavenes and chalcones may serve as candidates for skin-lightening agents.

Preliminary study on MC1R polymorphism in some cattle breeds raised in Italy

Most of the Western European cattle breeds consist of standardised breeds with a definite coat colour (Renieri et al., 1984). Thus coat colour could be useful to detect genetic markers for cattle breed identification. In cattle the pigmentation is determined by the distribution of two pigments: eu- and pheomelanin, producing brown or black and red to yellow pigmentation respectively. Tyrosinase, the rate-limiting enzyme involved in the synthesis of both melanins, is regulated by the melanocyte stimulating hormone (αMSH). This hormone and several other melanotropic peptides stimulate melanin formation in melanocytes by binding to the melanocortin-1-receptor (MC1R), a G-protein-coupled receptor encoded by the Extension gene (Robbins et al., 1993). In addition, the amounts of eu- and pheomelanin in the melanocyte are controlled by the agouti gene encoding the Agouti Signal Protein (ASP), that acts as an antagonist of MSH signalling through the MC1R, even if its mechanism of action is controversial (Furumura et al., 1998)

Latanoprost as a new horizon in the medical management of glaucoma

Latanoprost is a new prostaglandin F2 alpha analogue specifically developed for the treatment of glaucoma. Latanoprost is a selective FP receptor agonist, with a primary mode of action of increased uveoscleral outflow of aqueous humor. A dose of 50 micrograms/mL (0.005%) once daily has been found optimal in clinical trials. Latanoprost reduces the nocturnal intraocular pressure in addition to the diurnal, and has been shown to be additive to other glaucoma medication. In long-term phase III clinical trials, latanoprost 0.005% once daily has been proven to be at least as effective as timolol 0.5% twice a day. The main side effect of latanoprost is increased iridial pigmentation, which is relatively frequent in patients with mixed color of the iris. This unique side effect is based on the ability of prostaglandins to stimulate melanin formation in melanocytes. The advantages of latanoprost compared with other glaucoma medication comprise different mode of action, good intraocular pressure-reducing effect, once-daily dosing, and absence of systemic side effects. The long-term consequences of increased iridial pigmentation need to be further studied.

メラニン生成機構にかんする2，3の問題

The recent availability of cell biochemical techniques as ultracentrifugation separation of cell particles and density gradient centrifugation made possible the microdissection of and biochemical studies on melanin-forming cells which have elucidated the steps involved in the formation of melanosomes. A series of experiments along the studies on melanogenesis has been carried out within the last several years. Experimental data obtained from these studies are all compartible with the hypothesis, melanosome concept that tyrosinase is synthesized in ribosomes, transferred via endoplasmic reticulum to the Golgi area, where tyrosinase is separated into small units, each of which is surrounded by a membranous envelope. Within each envelope, the tyrosinase molecules assume an ordered pattern, after which melanin biosynthesis begins and the particle is known as a melanosome. As melanin gradually accumulates, the melanosome is transformed into a uniformly dense and structureless particle, a mature melanosome. The problems described herein are the in vitro experiments on the melanization process of melanosomes and the controling mechanism of thiol-group in melanin formation in melanocyte. The reciprocal relationship between tyrosinase activity and melanization of melanosomes has been demonstrated and data presented herein would be additional evidence to support this concept. Two kinds of melanosomes in defferent developmental stages were prepared from the mouse melanoma. Melanosome No. 1 is less melanized and possesses more tyrosinase activity than melanosome No. 2. Comparison of the rate of incorporation of 14C-DOPA in vitro by melanosome No. 1 and No. 2 was carried out. The experimental results showed that melanosome No. 1, immature melanosome can synthesize and deposit more melanin than melanosome No. 2, mature melanosomes. The melanization mechanism of melanosomes was further studied. Melanosomes isolated from the mouse melanoma were incubated with the excess amount of 14C-DOPA. The amount of melanin formed was dependent on the amount of melanosomes incubated but relationship between them was an upward convex rather than a straight line. It was assumed that there are some kinds of mechanisms which inactivate the enzyme. Kinetic studies were carried out in order to analyze the kind of inactivation mechanisms thought to be present. The exprimental results obtained could not be explained by assuming only one type of inactive enzymeproduct complex. However, they could be explained on the basis of simultaneous formation of two or more types of inactive enzymn-product complex. The sulfhydryl group has been considered as one of the important controling factors in melanin formation in melanocytes. The inhibitory effects of thiol compounds ont yrosine hydroxylase and tyrosinase in melanosomes isolated from mouse melanoma are descrided and an in vitro study of the reaction between an oxidation product of DOPA and glutathione in the presence of mammalian tyrosinase is reported. The formation of new chemical compounds resulted from the presence of DOPA, glutathione and mammalian tyrosinase in solution at pH 6.8. The existence of chemical bonding between glutathione and an oxidation product of DOPA, probably DOPA-quinone was demonstrated. The glutathione-derived reaction product remained soluble in the reaction medium and did not precipitate on the melanosomes. The pKi values of glutathione and cysteine on tyrosine hydroxylase were much smaller than those on tyrosinase. The inhibition of tyrosine melanin formation by sulfhydryl compounds may take place in two ways: by inhibition of the enzyme tyrosinase, and by the formation of new compounds in which intermediates of the tyrosine-tyrosinase reaction are trapped by the sulfhydryl compounds. (to be cotinued)

ON THE SITE OF MELANIN FORMATION IN MELANOCYTES.

Journal Article On the Site of Melanin Formation in Melanocytes Get access MAKOTO SEIJI, MAKOTO SEIJI Departments of Dermatology and Biochemistry Juntendo University School of MedicineBunkyo-ku, Tokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar SADAO IWASHITA SADAO IWASHITA Departments of Dermatology and Biochemistry Juntendo University School of MedicineBunkyo-ku, Tokyo Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 54, Issue 5, November 1963, Pages 465–467, https://doi.org/10.1093/oxfordjournals.jbchem.a127816 Published: 01 November 1963 Article history Received: 02 September 1963 Published: 01 November 1963