Melanin Synthesis In Melanocytes Research Articles

Commentary 5

Studies on the pathogenesis focussing on melanocyte biology, melanin biochemistry or skin immunity have yielded a variety of hypotheses to explain the disappearance of melanocytes in vitiligo, including a genetic predisposition, increased oxidative stress and toxic metabolites, neurochemical factors and autoimmunity (1). Whereas these factors probably all contribute to the development of depigmentation (convergence theory) (2), their interaction is not clearly defined. This commentary presents a multidisciplinary view of the pathogenesis of vitiligo (Fig. 1). Schematic overview of the pathogenesis of vitiligo. Melanin synthesis in melanocytes is a tightly regulated process. Defects in the protective mechanism in the skin that scavenge radicals and toxic intermediates of melanin production may lead to vitiligo. The increased levels of oxidative stress in the vitiligo skin leads to the deactivation of catalase (3). H202 can also oxidize (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin to 6-biopterin, which is cytotoxic to melanocytes. Although this melanocyte death may account for part of the depigmentation, dying melanocyte fragments will be taken up by epidermal Langerhans cells. The uptake of self-antigens from dying melanocytes in the absence of activation signals does not activate the Langerhans cells and will therefore not lead to immunity against melanocytes. Concurrent external stress factors, such as wounding, high dose of UV radiation or hormonal changes, however, activate Langerhans cells and dendritic cells in the skin, leading to breakage of tolerance (4). Clinically, these stress factors induce progression of depigmentation. The Koebner phenomenon results from stress factors at remote skin sites that induce local activation of Langerhans cells and reactivation of anti-melanocyte immunity, leading to the formation of new lesions. The lower threshold for breakage of tolerance in patients with vitiligo is illustrated by the increased incidence of autoimmune diseases in patients with vitiligo (5-12). Normally, regulatory T cells maintain peripheral tolerance by suppressing autoreactive T-cell activity. In patients with vitiligo, however, decreased levels of regulatory T cells in the skin lower the threshold for autoimmunity. Activated Langerhans cells containing melanocyte antigens migrate to the lymph nodes and present melanocyte antigenic peptides bound to HLA molecules to T cells. The association of vitiligo vulgaris with certain HLA class II alleles (13-15) indicates the dominance of peptides binding to these HLA types in inducing immunity. In the lymph nodes, melanocyte-reactive CD8+ and CD4+ T cells are activated to proliferate and migrate to the skin, resulting in increased levels of T cells reactive with tyrosinase, gp100 or MART-1 in peripheral blood (16-19), as well as autoantibodies against these antigens (20-22). Moreover, the number of circulating MART-1-reactive T cells expressing CLA in patients with vitiligo correlated with the extent of depigmentation (18). In perilesional skin, CD8+ T cells, macrophages and to a lesser extent CD4+ T cells were found (23, 24). Infiltrating CD8+ T cells expressed skin homing, cutaneous leukocyte-associated antigen (CLA) and T-cell activation markers CD25, perforin and granzyme B (25), and colocalized with disappearing melanocytes. Finally, perilesional melanocyte-reactive CD8+ cytotoxic T cells were shown to be capable of actively killing melanocytes in autologous skin tissue (J.G. van den Boorn, D. Konijnenberg, T.A.M. Dellemijn, J.P.W. van der Veen, J.D. Bos, C.J.M. Melief, F.A. Vyth-Dreese and R.M. Luiten, unpublished data). Taken together, the pathogenesis of vitiligo results from changes in biochemical processes in the skin that trigger autoimmunity, which is enhanced by genetic predisposition to autoimmunity.

Mechanism of the Inhibitory Effect of Tranexamic Acid on Melanogenesis in Cultured Human Melanocytes in the Presence of Keratinocyte-conditioned Medium

Tranexamic acid (TA) reduces hyperpigmentation in melasma patients, but the mechanism of its action is unknown. We have investigated the action of TA in human melanocyte cultures with or without keratinocyte-conditioned medium (KCM). In melanocyte cultures without KCM, TA in the concentration range of 0.5 mM to 5 mM did not reduce the activity of tyrosinase, a key enzyme of melanin synthesis, whereas it reduced the tyrosinase activity in the presence of KCM. These results indicate that TA inhibits melanin synthesis of melanocytes not by acting directly on melanocytes, but by inhibiting melanocyte activators contained in KCM. In fractionation studies of KCM, the stimulatory activity was predominantly contained in the fractions with an apparent molecular weight of 54000. Inhibition of the urokinase-type plasminogen activator (uPA) in KCM with specific anti-uPA antibody significantly decreased the KCM-induced increase of tyrosinase activity and inhibited the KCM-induced morphological changes of melanocytes. Our results suggest that TA inhibits melanin synthesis in melanocytes by interfering with the interaction of melanocytes and keratinocytes through inhibition of the plasminogen/plasmin system.

α-Melanocyte-stimulating Hormone Protects from Ultraviolet Radiation-induced Apoptosis and DNA Damage

Ultraviolet radiation is a well established epidemiologic risk factor for malignant melanoma. This observation has been linked to the relative resistance of normal melanocytes to ultraviolet B (UVB) radiation-induced apoptosis, which consequently leads to accumulation of UVB radiation-induced DNA lesions in melanocytes. Therefore, identification of physiologic factors regulating UVB radiation-induced apoptosis and DNA damage of melanocytes is of utmost biological importance. We show that the neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) blocks UVB radiation-induced apoptosis of normal human melanocytes in vitro. The anti-apoptotic activity of alpha-MSH is not mediated by filtering or by induction of melanin synthesis in melanocytes. alpha-MSH neither leads to changes in the cell cycle distribution nor induces alterations in the expression of the apoptosis-related proteins Bcl(2), Bcl(x), Bax, p53, CD95 (Fas/APO-1), and CD95L (FasL). In contrast, alpha-MSH markedly reduces the formation of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers, ultimately leading to reduced apoptosis. The reduction of UV radiation-induced DNA damage by alpha-MSH appears to be related to induction of nucleotide excision repair, because UV radiation-mediated apoptosis was not blocked by alpha-MSH in nucleotide excision repair-deficient fibroblasts. These data, for the first time, demonstrate regulation of UVB radiation-induced apoptosis of human melanocytes by a neuropeptide that is physiologically expressed within the epidermis. Apart from its ability to induce photoprotective melanin synthesis, alpha-MSH appears to exert the capacity to reduce UV radiation-induced DNA damage and, thus, may act as a potent protection factor against the harmful effects of UV radiation on the genomic stability of epidermal cells.

Tyrosinase Maturation and Oligomerization in the Endoplasmic Reticulum Require a Melanocyte-specific Factor

Tyrosinase is a glycoprotein responsible for the synthesis of melanin in melanocytes. A large number of mutations have been identified in tyrosinase, with many leading to its misfolding, endoplasmic reticulum (ER) retention, and degradation. Here we describe the folding and maturation of human tyrosinase (TYR) using an in vitro translation system coupled with ER-derived microsomes or with semipermeabilized cells, as an intact ER source. TYR remained misfolded as determined by its sensitivity to trypsin digestion and its persistent interaction with the ER resident lectin chaperones calnexin and calreticulin when produced in ER-derived microsomes or nonmelanocytic semipermeabilized cells. However, when TYR was translocated into semipermeabilized melanocytes, chaperone interactions were transient, maturation progressed to a trypsin-resistant state, and a TYR homodimer was formed. The use of semipermeabilized mouse melanocytes defective for tyrosinase or other melanocyte-specific proteins as the ER source indicated that proper TYR maturation and oligomerization were greatly aided by the presence of wild type tyrosinase and tyrosinase-related protein 1. These findings suggested that oligomerization is a step in proper TYR maturation within the ER that requires melanocyte-specific factors.

Hepatocyte growth factor/scatter factor-MET signaling in neural crest-derived melanocyte development.

The mechanisms governing development of neural crest-derived melanocytes, and how alterations in these pathways lead to hypopigmentation disorders, are not completely understood. Hepatocyte growth factor/scatter factor (HGF/SF) signaling through the tyrosine-kinase receptor, MET, is capable of promoting the proliferation, increasing the motility, and maintaining high tyrosinase activity and melanin synthesis of melanocytes in vitro. In addition, transgenic mice that ubiquitously overexpress HGF/SF demonstrate hyperpigmentation in the skin and leptomenigenes and develop melanomas. To investigate whether HGF/ SF-MET signaling is involved in the development of neural crest-derived melanocytes, transgenic embryos, ubiquitously overexpressing HGF/SF, were analyzed. In HGF/SF transgenic embryos, the distribution of melanoblasts along the characteristic migratory pathway was not affected. However, additional ectopically localized melanoblasts were also observed in the dorsal root ganglia and neural tube, as early as 11.5 days post coitus (p.c.). We utilized an in vitro neural crest culture assay to further explore the role of HGF/SF-MET signaling in neural crest development. HGF/SF added to neural crest cultures increased melanoblast number, permitted differentiation into pigmented melanocytes, promoted melanoblast survival, and could replace mast-cell growth factor/Steel factor (MGF) in explant cultures. To examine whether HGF/SF-MET signaling is required for the proper development of melanocytes, embryos with a targeted Met null mutation (Met-/-) were analysed. In Met-/- embryos, melanoblast number and location were not overtly affected up to 14 days p.c. These results demonstrate that HGF/SF-MET signaling influences, but is not required for, the initial development of neural crest-derived melanocytes in vivo and in vitro.

Histochemical and immunocytochemical ultrastructural localization of melanocyte specific proteins

Melanin synthesis in melanocytes is localized to melanosomes, where enzymatic and structural gene products must be targeted. Some gene products are translocated through different pathways (i.e., tyrosinase travels through the Golgi apparatus during processing, whereas a putative matrix glycoprotein does not). To visualize trafficking routes, we used post-embedding immunocytochemistry to compliment DOPA-histochemistry. We report on the localization of tyrosinase, matrix glycoprotein, tyrosinase-related protein 1 (TRP-1) and lysosome-associated membrane protein-1 (LAMP-1).DOPA histochemistry: Cultured melanoma cells were fixed in half-strength Karnovsky’s, incubated in 0.1 % 1-DOPA 2X for 2.5 h and embedded in Eponate 12. For immunocytochemistry, cells were fixed with 4% paraformaldehyde, 1% glutaraldehyde, and 0.2% picric acid with 0.5 mM CaCl2(pH 7.4) for 20 m at RT, quenched in 50 mM NH4Cl for 20 m at 0°C, washed in 0.1 M maleate buffer (pH 6.5), and treated with 2% uranyl acetate (pH 4.0) for 30 m at 0°C.

Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice.

The c-locus of the mouse is thought to encode tyrosinase, the key enzyme for melanin synthesis in melanocytes of the skin and the eye. Recently, a mouse cDNA was isolated and shown to confer tyrosine activity on a cell line which expressed no specialized functions for melanin synthesis. To verify that the isolated tyrosinase gene is encoded at the genetically well characterized c-locus, a minigene was assembled from tyrosinase cDNA and tyrosinase genomic DNA and used for generation of transgenic mice. Following microinjection of this construct into fertilized eggs of an albino mouse strain, transgenic mice were obtained which showed pigmentation in skin and eyes. By in situ hybridization, we show expression of the transgene in melanocytes of the hairbulb and in the pigmented cell layers of the eye. We conclude that we have rescued the albino mutation (c/c) by introduction and expression of a functional tyrosinase gene.

Regulation of Human Melanocyte Growth, Dendricity, and Melanization by Keratinocyte Derived Factors

Epidermal pigmentation involves the synthesis of melanin in melanocytes and its transfer to surrounding keratinocytes, where it functions in photoprotection. To investigate the possible role of the keratinocyte in regulating pigmentation, human keratinocytes were incubated for 24 h in a defined culture medium, which was then transferred to pure human melanocyte cultures. After 1 week, the conditioned medium produced a fourfold increase in melanocyte yield and a seven-fold increase in total melanin. Increased melanocyte dendricity was clearly visible within 24 h as well. Ultrafiltration of the keratinocyte-conditioned medium suggested approximately one-half of the growth promoting activity as well as most of the dendricity and melanization stimulating activities were of low molecular weight (less than 500 Da). High molecular weight fractions stimulated only melanocyte growth. Of the several known keratinocyte-derived factors tested, none could be implicated as a mediator of the observed effects. Basic fibroblast growth factor, known to stimulate melanocyte growth in some culture systems, failed to stimulate growth, dendricity, or melanin content when added to the complete non-conditioned medium. Interleukin-1 alpha, interleukin-1 beta, 12-hydroxyeicosatetraenoic acid, prostaglandin E2, leukotriene B4, and adenosine 3',5'-cyclic monophosphate analogues also had no effect. These studies demonstrate that keratinocytes in vitro release factors that modulate melanocyte behavior and expand our understanding of controls for human epidermal pigmentation.

Melanin-Related Metabolites as Markers of the Skin Pigmentary System

Three different groups of chemical intermediates are known to be formed during the synthesis of melanin in melanocytes: phenolic compounds, phenolic thio-conjugates, and indolic compounds. All these substances and their metabolites can be detected in urine. We measured the urinary excretion of 3,4-dihydroxyphenylalanine (dopa), 5-S-cysteinyldopa (5-S-CD), and 2 indolic compounds, namely 5-hydroxy-6-methoxyindole (5H6MI) and 5-hydroxy-6-methoxyindole-2-carboxylic acid (5H6MI2C) in urine samples of 4 groups of people with different contents of cutaneous melanin: Asian group, white group, and 2 groups of whites 1 with vitiligo and 1 with tyrosinase-negative oculocutaneous albinism. Dopa and 5-S-CD were determined with the method using high-performance liquid chromatography with an electrochemical detection. Indolic substances were measured by mass fragmentography with deuterium-labeled internal standards. Comparison of the melanin-related metabolites excreted in urine of people with different capacities for melanin biosynthesis indicates that, of all measured substances, 5H6MI2C is the best urinary marker of melanin formation in the skin pigmentary system.