Redefining the skin's pigmentary system with a novel tyrosinase assay

In mammalian skin, melanin is produced by melanocytes and transferred to epithelial cells, with the epithelial cells thought to receive pigment only and not generate it. Melanin formation requires the enzyme tyrosinase, which catalyzes multiple reactions in the melanin biosynthetic pathway. Here, we reassess cutaneous melanogenesis using tyramide-based tyrosinase assay (TTA), a simple test for tyrosinase activity in situ. In the TTA procedure, tyrosinase reacts with biotinyl tyramide, causing the substrate to deposit near the enzyme. These biotinylated deposits are then visualized with streptavidin conjugated to a fluorescent dye. In the skin and eye, TTA was highly specific for tyrosinase and served as a sensitive indicator of pigment cell distribution and status. In clinical skin samples, the assay detected pigment cell defects, such as melanocytic nevi and vitiligo, providing confirmation of medical diagnoses. In murine skin, TTA identified a new tyrosinase-positive cell type--the medullary cells of the hair--providing the first example of cutaneous epithelial cells with a melanogenic activity. Presumably, the epithelial tyrosinase originates in melanocytes and is acquired by medullary cells during pigment transfer. As tyrosinase by itself can generate pigment from tyrosine, it is likely that medullary cells produce melanin de novo. Thus, we propose that melanocytes convert medullary cells into pigment cells by transfer of the melanogenic apparatus, an unusual mechanism of differentiation that expands the skin's pigmentary system.     

Regulation of ornithine decarboxylase in B16 mouse melanoma cells: synergistic activation of melanogenesis by alphaMSH and ornithine decarboxylase inhibition

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines, a family of cationic compounds required for optimal cell proliferation and differentiation. Within mammalian melanocytes, the expression of genes regulating cell growth and/or differentiation can be controlled by alpha-melanocyte-stimulating hormone (alphaMSH) and other melanogenesis modulating agents. In the B16 mouse melanoma model, alphaMSH stimulates melanogenesis by upmodulation of tyrosinase (tyr) activity, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibits melanin synthesis. Therefore, we analyzed the regulation of ODC by these agents, as related to changes in the melanogenic pathway. Treatment of B16 cells with TPA or alphaMSH rapidly stimulated ODC activity. The effect was stronger for TPA and appeared mainly posttranslational. Irreversible inhibition of ODC with the active site-directed inhibitor alpha-difluoromethylornithine (DFMO) did not block TPA-mediated inhibition of tyr. Conversely, prolonged treatment of B16 cells with DFMO stimulated tyr activity by a posttranslational mechanism, probably requiring polyamine depletion. Combination treatment with alphaMSH and DFMO synergistically activated tyr. Therefore, ODC induction is not involved in the melanogenic response of B16 cells to alphaMSH. Rather, increased intracellular concentrations of polyamines following ODC induction might constitute a feedback mechanism to limit melanogenesis activation by alphaMSH.     

The Expression of Tyrosinase,Tyrosinase-Related Proteins 1 and 2 (TRP1 and TRP2), the Silver Protein,and a Melanogenic Inhibitor in Human Melanoma Cells of Differing Melanogenic Activities

The expression of various melanogenic proteins, including tyrosinase, the tyrosinase-related proteins 1 (TRP1) and 2 (TRP2/DOPAchrome tautomerase), and the silver protein in human melanocytes was studied in six different human melanoma cell lines and compared to a mouse derived melanoma cell line. Analysis of the expression of tyrosinase, TRP1, TRP2, and the silver protein using flow cytometry revealed that in general there was a positive correlation between melanin formation and the expression of those melanogenic enzymes. Although several of the melanoma cell lines possessed significant activities of TRP2, the levels of DOPAchrome tautomerase in extracts of human cells were relatively low compared to those in murine melanocytes. Melanins derived from melanotic murine JB/MS cells, from melanotic human Ihara cells and HM-IY cells, from sepia melanin, and from C57BL/6 mouse hair were chemically analyzed. JB/MS cells, as well as Ihara cells and HM-TY cells, possessed significant amounts of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derived melanins, this being dependent on the activity of TRP2. Kinetic HPLC assays showed that 5,6-dihydroxyindole (DHI) produced during melanogenesis was metabolized quickly to melanin in pigmented KHm-1/4 cells, whereas DHI was stable in amelanotic human SK-MEL-24 cells. A melanogenic inhibitor that has been purified from SK-MEL-24 cells that suppressed oxidation of DHI in the presence or absence of tyrosinase, but had no effect on DHICA oxidation. The sum of these results suggest that the expression of melanogenic enzymes as well as the activity of a melanogenic inhibitor are critical to the production of melanin synthesis in humans.     

Melanocyte subpopulation turnover during the human hair cycle: an immunohistochemical study

The human hair cycle is characterized by successive phases of growth and involution that imply tissue regression and regeneration. As a consequence, the hair melanin unit has to be renewed in a cyclic manner. Actually, the behavior of human hair follicle melanocytes throughout the hair cycle has been poorly studied. Thus, the origin of melanocytes present in the bulb after human hair regeneration is still not clarified, and neither are the events that control the melanin biosynthesis activity in the human hair bulb. In this study, we showed at the cellular level that in human pigmented hair follicles, the expression of tyrosinase and tyrosinase-related protein-1 (TRP-1) was detectable during the anagen phases III/IV through VI, only in those melanocytes which were located in the bulb. During the catagen phase, the two evaluated melanogenic enzymes were detectable no more, although melanocytes were still present in the preceding bulbar area. The epithelial column of catagen follicles and the capsule of telogen follicles also contained inactive melanocytes as evidenced by pMel-17 labeling. At the induction of a new anagen hair follicle, some melanocytes were committed to cell division, but only when located in the nascent bulb close to the dermal papilla. Our results emphasize the close relationship between melanogenesis and the hair cycle and suggest that in humans, melanogenesis is restricted to anagen hair follicles not because of the regulation of tyrosinase activity, but because of melanogenic enzyme expression, e.g., tyrosinase and TRP-1. Furthermore, the fact that in the newly developing anagen hair follicles, cell-division commitment and tyrosinase and TRP-1 expression were observed in melanocytes only when located in the nascent bulb suggests a highly regio-specific melanocyte stimulation in early the anagen phase.     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

Analysis of the UV-Induced Melanogenesis and Growth Arrest of Human Melanocytes

Cultured human melanocytes derived from different skin types responded to frequent treatment with ultraviolet (UV) light with increased melanin synthesis, decreased proliferation, and morphologic signs of aging. These effects were augmented by increased frequency of irradiation with 15.5 mJ/cm² UV light. Stimulation of melanogenesis by UV light involved an increase in tyrosinase activity, without any change in the amounts of either tyrosinase or tyrosinase-related protein (TRP)-1, and a decrease in the amount of TRP-2, as determined by Western blot analysis. These results are different from the mechanisms by which other melanogenic agents, such as cholera toxin and isobutyl methylxanthine, stimulated melanogenesis, whereby the amounts of tyrosinase, TRP-1 and TRP-2 were increased. The decrease in the amount of TRP-2 might be significant in that it might alter the properties of the newly synthesized melanin. The UV irradiation protocol that was followed blocked melanocytes in G₂-M phase of the cell cycle without compromising cellular viability. Following three rounds of UV irradiation, melanocytes could recover from the growth arrest and resume proliferation. Treatment with 0.1 μM α-melanocyte stimulating hormone (α-MSH) postirradiation enhanced the melanogenic effect of UV light and stimulated the melanocytes to proliferate. The effects of α-MSH on the UV induced responses and their implications on photocarcinogenesis are being further investigated. Analyzing the mechanisms by which UV light exposure affects normal melanocytes might lead to a better understanding of how these cells undergo malignant transformation, and why individuals with different skin types differ in their susceptibility to skin cancers.     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

Tyrosinase related protein 1 (TRP1) functions as a DHICA oxidase in melanin biosynthesis.

Several genes critical to the enzymatic regulation of melanin production in mammals have recently been cloned and mapped to the albino, brown and slaty loci in mice. All three genes encode proteins with similar structures and features, but with distinct catalytic capacities; the functions of two of those gene products have previously been identified. The albino locus encodes tyrosinase, an enzyme with three distinct melanogenic functions, while the slaty locus encodes tyrosinase-related protein 2 (TRP2), an enzyme with a single specific, but distinct, function as DOPAchrome tautomerase. Although the brown locus, encoding TRP1, was actually the first member of the tyrosinase gene family to be cloned, its catalytic function (which results in the production of black rather than brown melanin) has been in general dispute. In this study we have used two different techniques (expression of TRP1 in transfected fibroblasts and immunoaffinity purification of TRP1 from melanocytes) to examine the enzymatic function(s) of TRP1. The data demonstrate that the specific melanogenic function of TRP1 is the oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) to a carboxylated indole-quinone at a down-stream point in the melanin biosynthetic pathway. This enzyme activity appears to be essential to the further metabolism of DHICA to a high molecular weight pigmented biopolymer.     

Regulation of mammalian melanogenesis by tyrosinase inhibition

Melanocyte stimulating hormone (MSH) specifically induces differentiation of mammalian melanocytes. To further define the biochemical events elicited by this stimulus, we have cloned murine melanoma cells which are either highly responsive or nonresponsive to MSH, and have examined their ultrastructural appearance, their melanogenic activities, and also their expression of tyrosinase. We have found that the basal levels of melanogenic activity in pigmented and nonpigmented cells correlate with expression of surface MSH receptors rather than with production of tyrosinase. Nonpigmented cells produce a potent, highly stable inhibitor of melanogenesis; this inhibitor acts directly on tyrosinase to dramatically and abruptly suppress melanin production. This posttranslational control of tyrosinase activity may represent a critical regulatory point in mammalian pigmentation.     

Regulation of tyrosinase in human melanocytes grown in culture

Tyrosinase, the enzyme that controls the synthesis of melanin, is a unique product of melanocytes. Normal and malignant human melanocytes grown in culture were used to study the factors that regulate the expression of tyrosinase. Immunoprecipitation experiments showed that newly synthesized tyrosinase appeared as a protein with an apparent molecular weight of 70,000 that was processed to a protein with an apparent molecular weight of 80,000. Neither tunicamycin nor 2-deoxy-D- glucose inhibited this conversion, suggesting that O-glycosylation is the major biochemical event in the posttranslational modification of tyrosinase. Agents that stimulated the proliferation of normal melanocytes also stimulated tyrosinase activity. Melanocytes with low levels of tyrosinase activity synthesized less tyrosinase, processed the enzyme more slowly, and degraded it more rapidly than melanocytes with high levels of tyrosinase activity. We conclude that tyrosinase activity in cultures of human melanocytes derived from different donors is determined predominantly by its abundance.