Increased expression of integrins and decreased apoptosis correlate with increased melanocyte retention in cultured skin substitutes

Losses of human melanocytes (HM) in transplantation of cultured skin substitutes (CSS) may result from poor cellular attachments. To test this hypothesis, HM integrin expression was measured in four culture media: (a) melanocyte growth medium (MGM), an HM proliferation medium; (b) UCMC 160, a CSS maturation medium; (c) mMGM, modified MGM with 1.8 mM calcium; and (d) modified UCMC 160 with HM supplements (mUCMC 160). HM grew well in all media except UCMC 160. Increased expression of beta1, beta4, alpha3beta1 and alpha5 integrins on HM cultured in MGM and mMGM versus UCMC 160 was found by flow cytometry. Annexin V-allophycocyanin (APC) labeled HM in apoptosis and increased significantly in UCMC 160 (31.1%) compared with MGM (11.9%) or mMGM (13.9%). CSS were incubated in UCMC 160, mMGM or mUCMC 160 media, and grafted to athymic mice. In the mMGM group, grafts were darker as measured with a chromameter through 6 weeks and the average number of basal HM per field was greater at 12 weeks post-grafting. Increased graft loss was observed in the mMGM group which corresponded with the poor epidermal morphology in vitro. Although HM retention improved in vivo using mMGM to culture the CSS, the stability of the epidermis decreased. These results indicate that expression of integrins on HM in vitro correlates with HM retention in CSS and short-term survival after transplantation, but that long-term survival depends also on stable epithelium.     

The deceptive nature of UVA tanning versus the modest protective effects of UVB tanning on human skin

The relationship between human skin pigmentation and protection from ultraviolet (UV) radiation is an important element underlying differences in skin carcinogenesis rates. The association between UV damage and the risk of skin cancer is clear, yet a strategic balance in exposure to UV needs to be met. Dark skin is protected from UV-induced DNA damage significantly more than light skin owing to the constitutively higher pigmentation, but an as yet unresolved and important question is what photoprotective benefit, if any, is afforded by facultative pigmentation (i.e. a tan induced by UV exposure). To address that and to compare the effects of various wavelengths of UV, we repetitively exposed human skin to suberythemal doses of UVA and/or UVB over 2 weeks after which a challenge dose of UVA and UVB was given. Although visual skin pigmentation (tanning) elicited by different UV exposure protocols was similar, the melanin content and UV-protective effects against DNA damage in UVB-tanned skin (but not in UVA-tanned skin) were significantly higher. UVA-induced tans seem to result from the photooxidation of existing melanin and its precursors with some redistribution of pigment granules, while UVB stimulates melanocytes to up-regulate melanin synthesis and increases pigmentation coverage, effects that are synergistically stimulated in UVA and UVB-exposed skin. Thus, UVA tanning contributes essentially no photoprotection, although all types of UV-induced tanning result in DNA and cellular damage, which can eventually lead to photocarcinogenesis.     

Anti-melanogenesis potential of a new series of Morita-Baylis-Hillman adducts in B16F10 melanoma cell line

Melanin is a natural polymer pigment which provides skin photoprotection against ultraviolet radiation. An excessive synthesis of melanin leads to hyperpigmentation disorders. Tyrosinase catalyzes the rate limiting steps on melanogenesis. Therefore, tyrosinase inhibitors have potential applications in medicine and cosmetic fields. We carried out herein the screening of a family of cyclic Morita-Baylis-Hillman adducts (MBH) to find out their effects on tyrosinase activity and on melanogenesis in murine melanoma B16F10 cell line. Kinetic analysis of tyrosinase inhibition showed that compounds 1a (2-hydroxymethyl) cyclohex-2-enone) and 3f (diethyl (1-(6-oxocyclohex-1-en-1-yl) ethyl-phosphonate) were competitive inhibitors, whereas the compound 2b (6-oxocyclohex-1-en-1-yl) ethyl acetate) was a non-competitive one. Additionally we have found that (1a, 2b and 3f) compounds had a strong melanogenesis inhibition effect in isobutylmethylxanthine (IBMX)-treated murine melanoma B16F10 cells when tested at low and non cytotoxic dose (10–50 µM), by attenuating the melanin production, intracellular tyrosinase activity and tyrosinase expression. Thus, we suggest that these compounds could be used as effective skin-whitening agents.     

Comparison of Long‐Term Survival of Pigmented Epidermal Reconstructs Cultured In Vitro vs. Xenografted on Nude Mice

Epidermal reconstructs incorporating pigment cells have been used in vitro over the last decade to study the physiology of the epidermal melanin unit. However, the major limitation of this technology is the duration of the assays, which need to be completed within 2–3 weeks to obviate the problem of epidermal senescence and excessive terminal differentiation. This becomes a major problem for studying long‐term biological phenomena in photoprotection and epidermal skin cancers. We report here a simplified surgical technique in immunotolerant mice allowing long‐term studies. The creation of a vascularized mouse skin flap is the key point of the surgical procedure. Long‐term pigmentation of the xenografts seemed macroscopically successful, but surprisingly microscopy at 11 and 16 weeks postgrafting showed mostly dermal pigment aggregates and rare Melan‐A positive dermal and epidermal pigment cells. In the same reconstructs maintained in vitro, dermal pigment and dermal pigment cells were never noted. It could be speculated that in our model, the colonization of the xenografted dead human dermis by murine cells influences melanocyte survival.     

Arginase‐2, a miR‐1299 target,enhances pigmentation in melasma by reducing melanosome degradation via senescence‐induced autophagy inhibition

Expression profiles revealed miR‐1299 downregulation concomitant with arginase‐2 (ARG2) upregulation in hyperpigmented skin of melasma patients. Opposite regulation of tyrosinase and PMEL17 by miR‐1299 and inverse relationship between miR‐1299 and ARG2 expression denoted a role of miR‐1299 in pigmentation with ARG2 as a miR‐1299 target. ARG2 overexpression or knock‐down in keratinocytes, the main source of ARG2 in epidermis, positively regulated tyrosinase and PMEL17 protein levels, but not mRNA levels or melanosome transfer. ARG2 overexpression in keratinocytes reduced autophagy equivalent to 3‐MA, an autophagy inhibitor which also increased tyrosinase and PMEL17 protein levels, whereas ARG2 knock‐down induced opposite results. Autophagy inducer rapamycin reduced ARG2‐increased tyrosinase and PMEL17 protein levels. Also, autophagy was reduced in late passage‐induced senescent keratinocytes showing ARG2 upregulation. ARG2, but not 3‐MA, stimulated keratinocyte senescence. These results suggest that ARG2 reduces autophagy in keratinocytes by stimulating cellular senescence, resulting in skin pigmentation by reducing degradation of transferred melanosomes.     

Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors

Melanogenesis is a process to synthesize melanin, which is a primary responsible for the pigmentation of human skin, eye and hair. Although numerous enzymatic catalyzed and chemical reactions are involved in melanogenesis process, the enzymes such as tyrosinase and tyrosinase-related protein-1 (TRP-1) and TRP-2 played a major role in melanin synthesis. Specifically, tyrosinase is a key enzyme, which catalyzes a rate-limiting step of the melanin synthesis, and the downregulation of tyrosinase is the most prominent approach for the development of melanogenesis inhibitors. Therefore, numerous inhibitors that target tyrosinase have been developed in recent years. The review focuses on the recent discovery of tyrosinase inhibitors that are directly involved in the inhibition of tyrosinase catalytic activity and functionality from all sources, including laboratory synthetic methods, natural products, virtual screening and structure-based molecular docking studies.     

The TYRP1‐mediated protection of human tyrosinase activity does not involve stable interactions of tyrosinase domains

Tyrosinases are melanocyte‐specific enzymes involved in melanin biosynthesis. Mutations in their genes cause oculocutaneous albinism associated with reduced or altered pigmentation of skin, hair, and eyes. Here, the recombinant human intra‐melanosomal domains of tyrosinase, TYRtr (19–469), and tyrosinase‐related protein 1, TYRP1tr (25–472), were studied in vitro to define their functional relationship. Proteins were expressed or coexpressed in whole Trichoplusia ni larvae and purified. Their associations were studied using gel filtration and sedimentation equilibrium methods. Protection of TYRtr was studied by measuring the kinetics of tyrosinase diphenol oxidase activity in the presence (1:1 and 1:20 molar ratios) or the absence of TYRP1tr for 10 hr under conditions mimicking melanosomal and ER pH values. Our data indicate that TYRtr incubation with excess TYRP1tr protects TYR, increasing its stability over time. However, this mechanism does not appear to involve the formation of stable hetero‐oligomeric complexes to maintain the protective function.     

Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio

The skin constitutive pigmentation is given by the amount of melanin pigment, its relative composition (eu/pheomelanin) and distribution within the epidermis, and is largely responsible for the sensitivity to UV exposure. Nevertheless, a precise knowledge of melanins in human skin is lacking. We characterized the melanin content of human breast skin samples with variable pigmentations rigorously classified through the Individual Typology Angle (ITA) by image analysis, spectrophotometry after solubilization with Soluene‐350 and high‐performance liquid chromatography (HPLC) after chemical degradation. ITA and total melanin content were found correlated, ITA and PTCA (degradation product of DHICA melanin), and TTCA (degradation product of benzothiazole‐type pheomelanin) as well but not 4‐AHP (degradation product of benzothiazine‐type pheomelanin). Results revealed that human epidermis comprises approximately 74% of eumelanin and 26% pheomelanin, regardless of the degree of pigmentation. They also confirm the low content of photoprotective eumelanin among lighter skins thereby explaining the higher sensitivity toward UV exposure.     

Screening of Human Primary Melanocytes of Defined Melanocortin‐1 Receptor Genotype: Pigmentation Marker,Ultrastructural and UV‐Survival Studies

Recent population studies have demonstrated an association with the red‐hair and fair‐skin phenotype with variant alleles of the melanocortin‐1 receptor (MC1R) which result in amino acid substitutions within the coding region leading to an altered receptor activity. In particular, Arg151Cys, Arg160Trp and Asp294His were the most commonly associated variants seen in the south‐east Queensland population with at least one of these alleles found in 93% of those with red hair. In order to study the individual effects of these variants on melanocyte biology and melanocytic pigmentation, we established a series of human melanocyte strains genotyped for the MC1R receptor which included wild‐type consensus, variant heterozygotes, compound heterozygotes and homozygotes for Arg151Cys, Arg160Trp, Val60Leu and Val92Met alleles. These strains ranged from darkly pigmented to amelanotic, with all strains of consensus sequence having dark pigmentation. UV sensitivity was found not to be associated with either MC1R genotype or the level of pigmentation with a range of sensitivities seen across all genotypes. Ultrastructural analysis demonstrated that while consensus strains contained stage IV melanosomes in their terminal dendrites, Arg151Cys and Arg160Trp homozygote strains contained only stage II melanosomes. This was despite being able to show expression of tyrosinase and tyrosinase‐related protein‐1 markers, although at reduced levels and an ability to convert exogenous 3,4‐dihydroxyphenyl‐alanine (DOPA) to melanin in these strains.     

Influence of Temperature of Incubation and Type of Growth Medium on Pigmentation in Serratia marcescens

Maximal amounts of prodigiosin were synthesized in either minimal or complete medium after incubation of cultures at 27 C for 7 days. Biosynthesis of prodigiosin began earlier and the range of temperature for formation was greater in complete medium. No prodigiosin was formed in either medium when cultures were incubated at 38 C; however, after a shift to 27 C, pigmentation ensued, provided the period of incubation at 38 C was not longer than 36 hr for minimal medium or 48 hr for complete medium. Washed, nonpigmented cells grown in either medium at 38 C for 72 hr could synthesize prodigiosin when suspended in saline at 27 C when casein hydrolysate was added. These suspensions produced less prodigiosin at a slower rate than did cultures growing in casein hydrolysate at 27 C without prior incubation at 38 C. Optimal concentration of casein hydrolysate for pigment formation by suspensions was 0.4%; optimal temperature was 27 C. Anaerobic incubation, shift back to 38 C, killing cells by heating, or chloramphenicol (25 mug/ml) inhibited pigmentation. Suspensions of washed cells forming pigment reached pH 8.0 to 8.3 rapidly and maintained this pH throughout incubation for 7 days. Measurements of viable count and of protein, plus other data, indicated that cellular multiplication did not occur in suspensions of washed cells during pigment formation. By this procedure utilizing a shift down in temperature, biosynthesis of prodigiosin by washed cells could be separated from multiplication of bacteria.     

Lightening Effect on Ultraviolet‐Induced Pigmentation of Guinea Pig Skin by Oral Administration of a Proanthocyanidin‐Rich Extract from Grape Seeds

Antioxidants such as vitamins C and E have been reported to inhibit the progression of ultraviolet (UV) radiation‐induced pigmentation in the skin of hairless mice. However, little is known of the lightening effect of proanthocyanidin, a powerful polyphenolic antioxidant, on UV‐induced pigmentation of the skin. We investigated the lightening effect of oral administration of a proanthocyanidin‐rich grape seed extract (GSE) using guinea pigs with UV‐induced pigmentation. These pigmented guinea pigs were fed diets containing 1% GSE or 1% vitamin C (w/w) for 8 weeks. GSE‐feeding had an apparent lightening effect on the guinea pigs’ pigmented skin. Histologic evaluation demonstrated a decrease in the number of 3,4‐dihydroxyphenylalanine (DOPA)‐positive melanocytes as well as 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG)‐positive, Ki‐67‐positive, proliferating cell nuclear antigen (PCNA)‐positive melanin‐containing cells in the basal epidermal layer of the UV‐irradiated skin in GSE‐fed guinea pigs. In contrast, these parameters did not change in the skin of vitamin C‐fed or control guinea pigs. GSE inhibited the activity of mushroom tyrosinase and also inhibited melanogenesis without inhibiting the growth of cultured B16 mouse melanoma cells. In conclusion, we demonstrated that oral administration of GSE is effective in lightening the UV‐induced pigmentation of guinea pig skin. This effect may be related to the inhibition of melanin synthesis by tyrosinase in melanocytes and the reactive oxygen species (ROS)‐related proliferation of melanocytes.     

Melanogenesis and Melanoma

Melanins are the principal surface pigments in vertebrates and, in humans, play a major role in photoprotection. Although the product (melanin) has a mainly protective function in the skin, the process of melanogenesis represents a potential cellular hazard and is confined to special membrane‐limited organelles (melanosomes) in a set of specialized dendritic cells (melanocytes) which synthesize the pigment and transfer it to recipient cells. Malignant melanocytes tend to exhibit up‐regulated melanogenesis and defective melanosomes. These features suggest ways in which anti‐melanoma therapy may be specifically targeted. Two general chemotherapeutic modalities are considered:
1 The ‘Achilles heel’ approach in which the generation of reactive quinones capable of leaking into the cytosolic compartment and causing structural and functional derangement is encouraged by the use of analogue substrates.
2 The ‘Trojan horse’ approach, in which a cytotoxic agent is selectively released by a tyrosinase‐dependent mechanism.     

Melanin processing by keratinocytes: A non‐microbial type of host‐pathogen interaction?

The mechanisms that regulate skin pigmentation have been the subject of intense research in recent decades. In contrast with melanin biogenesis and transport within melanocytes, little is known about how melanin is transferred and processed within keratinocytes. Several models have been proposed for how melanin is transferred, with strong evidence supporting coupled exo/endocytosis. Recently, two reports suggest that upon internalization, melanin is stored within keratinocytes in an arrested compartment, allowing the pigment to persist for long periods. In this commentary, we identify a striking parallelism between melanin processing within keratinocytes and the host‐pathogen interaction with Plasmodium, opening new avenues to understand the complex molecular mechanisms that ensure skin pigmentation and photoprotection.      

Regulation of eumelanin/pheomelanin synthesis and visible pigmentation in melanocytes by ligands of the melanocortin 1 receptor

The production of melanin in the hair and skin is tightly regulated by the melanocortin 1 receptor (MC1R) whose activation is controlled by two secreted ligands, alpha-melanocyte stimulating hormone (alphaMSH) and agouti signal protein (ASP). As melanin is extremely stable, lasting years in biological tissues, the mechanism underlying the relatively rapid decrease in visible pigmentation elicited by ASP is of obvious interest. In this study, the effects of ASP and alphaMSH on the regulation of melanin synthesis and on visible pigmentation were assessed in normal murine melanocytes and were compared with the quick depigmenting effect of the tyrosinase inhibitor, phenylthiourea (PTU). alphaMSH increased pheomelanin levels prior to increasing eumelanin content over 4 days of treatment. Conversely, ASP switched off the pigment synthesis pathway, reducing eu- and pheo-melanin synthesis within 1 day of treatment that was proportional to the decrease in tyrosinase protein level and activity. These results demonstrate that the visible depigmentation of melanocytes induced by ASP does not require the degradation of existing melanin but rather is due to the dilution of existing melanin by melanocyte turnover, which emphasizes the importance of pigment distribution to visible color.     

Human hair melanins: what we have learned and have not learned from mouse coat color pigmentation

Hair pigmentation is one of the most conspicuous phenotypes in humans. Melanocytes produce two distinct types of melanin pigment: brown to black, indolic eumelanin and yellow to reddish brown, sulfur‐containing pheomelanin. Biochemically, the precursor tyrosine and the key enzyme tyrosinase and the tyrosinase‐related proteins are involved in eumelanogenesis, while only the additional presence of cysteine is necessary for pheomelanogenesis. Other important proteins involved in melanogenesis include P protein, MATP protein, α‐MSH, agouti signaling protein (ASIP), MC1R (the receptor for MSH and ASIP), and SLC7A11, a cystine transporter. Many studies have examined the effects of loss‐of‐function mutations of those proteins on mouse coat color pigmentation. In contrast, much less is known regarding the effects of mutations of the corresponding proteins on human hair pigmentation except for MC1R polymorphisms that lead to pheomelanogenesis. This perspective will discuss what we have/have not learned from mouse coat color pigmentation, with special emphasis on the significant roles of pH and the level of cysteine in melanosomes in controlling melanogenesis. Based on these data, a hypothesis is proposed to explain the diversity of human hair pigmentation.     

Influence of tyrosinase levels on pigment accumulation in the retinal pigment epithelium and on the uncrossed retinal projection

To study the relationship among tyrosinase activity, melanin production, and the routing of retinal ganglion cell (RGC) axons at the optic chiasm, we analysed mice with varying doses of the tyrosinase gene. These include the dark-eyed albino (Tyrc44H), a radiation-induced hypomorphic allele of tyrosinase; and transgenic mice carrying 1 or 2 alleles of a tyrosinase minigene on both wild-type (Tyr+) and albino (Tyrc) backgrounds. Melanization of the retinal pigment epithelium (RPE) occurred gradually even at <2% wild-type tyrosinase activity and was sensitive to tyrosinase activity up to <35% of wild-type levels, beyond which melanin synthesis appeared to be saturated. Overexpression of tyrosinase led to tyrosinase activity above wild type level, but did not increase melanin production. Although a loss of melanin because of a mutation in tyrosinase is associated with a decrease in the number of uncrossed fibers, elevating tyrosinase levels does not appear to cause an increase in the size of the uncrossed retinal projection. Our results suggest that replacing less than 35% of wild-type tyrosinase activity is sufficient to restore normal pigmentation of the RPE, and potentially, to allay visual defects.     

Analogues of N-hydroxy-N'-phenylthiourea and N-hydroxy-N'-phenylurea as inhibitors of tyrosinase and melanin formation

A series of N-hydroxy-N'-phenylthiourea and N-hydroxy-N'-phenylurea analogues were prepared and evaluated as inhibitors of tyrosinase and melanin formation. The most active analogue 1 inhibited mushroom tyrosinase with an IC(50) of around 0.29 microM and also retained a substantial potency in cell culture by reducing pigment synthesis by 78%. Therefore, compound 1 could be considered as a promising candidate for preclinical drug development for skin hyperpigmentation application.