Tyrosinase in the presumptive pigment cells of ascidian embryos: Tyrosine accessibility may initiate melanin synthesis

Tyrosinase activity appears in the presumptive pigment cells of ascidian embryos (Ciona intestinalis) several hours before the cells begin to synthesize melanin. These presumptive pigment cells develop into the otolith and ocellus pigment cells of the larval brain. Tyrosinase was identified by histochemical tests for tyrosine oxidase and dopa oxidase; both reactions were sensitive to tyrosinase inhibitors. Studies with puromycin suggested that tyrosinase was synthesized at the time it was first detected histochemically and that it was stable during the time interval before melanin synthesis. Supernumerary tyrosinase-containing cells were found adjacent to the presumptive pigment cells in three ascidian species examined (C. intestinalis, Styela partita, and Molgula manhattensis). Tyrosinase disappeared from the supernumerary pigment cells during larval development and these cells did not synthesize melanin.Tyrosinase in the presumptive and supernumerary pigment cells is apparently a functional enzyme which does not interact with substrate. External substrates ( -tyrosine and -dopa) did not react with enzyme in the living cells before the normal time of pigment synthesis, but gentle disruption of the cells (by freezing-and-thawing or osmotic shock) released active tyrosinase. Progessive enlargement of nonpigmented vesicles in the otolith cells of embryos exposed to phenylthiourea, an inhibitor of tyrosinase activity, suggested that tyrosinase vesicles actively accumulate tyrosine at the beginning of melanin synthesis. This tyrosine accumulation probably initiates melanin synthesis.     

Occurrence of melanin granules and melanosynthesis in the kidney of Sparus auratus

An optical, ultrastructural, and biochemical study of the melanin accumulation nodules found in the kidney of the teleost fish Sparus auratus is presented. These nodules are randomly distributed in the interstitium of the renal tissue. They are formed by large aggregates of cells replete with melanin granules. The melanin granules occur singly or also in aggregates inside the cells. Most of the granules are electron-dense, but sometimes small electron-lucent spaces within them can be seen. Some secondary lysosomes and dendritic processes can also be observed. Biochemical studies have proved for the first time the existence of measurable tyrosinase activity in those nodules. That activity was assayed using three methods: tyrosine hydroxylation, dopa oxidation, and melanin formation. Furthermore, inhibitors of well-characterized plant and animal skin tyrosinases were effective agents for inhibiting those activities in fish kidney preparations. This finding supports the notion of the existence of true tyrosinase in the melanin accumulation nodules of this tissue. Taking into account the results obtained, the origin and functions of the melanin-containing cells found in the teleost fish kidney are discussed.     

Natural,semisynthetic and synthetic tyrosinase inhibitors

Tyrosinase plays a pivotal role in the synthesis of melanin pigment synthesis on skin utilizing tyrosine as a substrate. Melanin is responsible for the protection against harmful ultraviolet irradiation, which can cause significant pathological conditions, such as skin cancers. However, it can also create esthetic problems when accumulated as hyperpigmented spots. Various skin-whitening ingredients which inhibit tyrosinase activity have been identified. Some of them, especially ones with natural product origins, possess phenolic moiety and have been employed in cosmetic products. Semi-synthetic and synthetic inhibitors have also been developed under inspiration of the natural inhibitors yet some of which have no phenolic groups. In this review, tyrosinase inhibitors with natural, semi-synthetic and synthetic origins are listed up with their structures, activities and characteristics. Further, a recent report on the adverse effect of a natural melanin synthesis inhibitor which was included in skin-whitening cosmetics is also briefly discussed.     

A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase.

The production of melanin pigment in mammals requires tyrosinase, an enzyme which hydroxylates the amino acid tyrosine to DOPA (3,4-dihydroxyphenylalanine), thus allowing the cascade of reactions necessary to synthesize that biopolymer. However, there are other regulatory steps that follow the action of tyrosinase and modulate the quantity and quality of the melanin produced. DOPAchrome tautomerase is one such melanogenic enzyme that isomerizes the pigmented intermediate DOPAchrome to DHICA (5,6-dihydroxyindole-2-carboxylic acid) rather than to DHI (5,6-dihydroxyindole), which would be generated spontaneously. This enzyme thus regulates a switch that controls the proportion of carboxylated subunits in the melanin biopolymer. Efforts to clone the gene for tyrosinase have resulted in the isolation of a family of tyrosinase related genes which have significant homology and encode proteins with similar predicted structural characteristics. Using specific antibodies generated against synthetic peptides encoded by unique areas of several of those proteins, we have immuno-affinity purified them and studied their melanogenic catalytic functions. We now report that TRP-2 (tyrosinase related protein-2), which maps to and is mutated at the slaty locus in mice, encodes a protein with DOPAchrome tautomerase activity.     

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.     

Chromogenesis mirabilis in Streptomyces griseus

A number of chromogenic Streptomyces, producing diffusible melanoid pigment on complex organic media, fail to form melanin pigment on conventionally used synthetic tyrosine agar. By means of our new melanin formation test, almost all the chromogenic streptomyces can now be detected in chemically defined medium. In contrast to ordinary chromogenic streptomyces, two streptomyces species of the International Streptomyces Project, S. griseus ISP 5236 and S. ornatus ISP 5307, produce melanin pigment only on synthetic tyrosine agar, without showing chromogenicity on complex organic media. From the results obtained with S. griseus ISP 5236 and S. phaeochromogenes ISP 5073, it was revealed that melanin formation by Streptomyces, in general, is inhibited by L-cysteine present in organic nitrogen sources incorporated into natural media. Most chromogenic species of streptomyces produce a higher level of tyrosinase and rapidly utilize L-cysteine in the culture media which result in the manifestation of good chromogenicity on natural media. Peculiarity of chromogenicity of S. griseus and S. ornatus might be due to the lower ability to produce tyrosinase and to utilize L-cysteine in the culture medium.     

THE DIFFERENTIATION OF PIGMENT CELLS IN CULTURES OF CHICK EMBRYONIC NEURAL RETINAE

Neural retinal cells of 8–9 day-old chick embryos were differentiated into pigment cells in the conditions of cell culture for about 25 days. The increase of pigment cells in vitro was semi-quantitatively shown, by counting the number of black foci of pigmented cells per plate throughout the culture period. The increase paralleled the increase in the activity of tyrosinase. The addition of a small number of pigment cells freshly dissociated from tapeta to the cultures of neural retinae did not increase the number of black foci in vitro . Electron microscopic observations revealed the morphological differences of melanin granules between those in pigment cells of the neural retinal cultures and those in cultured tapetum cells. It was discussed that pigment cells appearing in the neural retinal cultures were derived from neural retinal cells, but not from contaminated cells of the tapetum.     

Tumor Suppressor p53 Down-Regulates Tissue-Specific Expression of Tyrosinase Gene in Human Melanoma Cell Lines

Tyrosinase, the key gene in melanin pigment synthesis, is tissue-specifically expressed in melanocytic cells. Expression of this gene is regulated by various hormones, carcinogens, and environmental factors. The molecular basis underlying tyrosinase gene regulation is still not clear. In this report, we present the effects of tumor suppressor p53 protein on tyrosinase gene expression and melanin synthesis in human melanoma. After stable transfection of wild type p53 expression plasmid into a highly pigmented melanoma cell line, overexpression of wt p53 suppressed the pigmentation of the melanoma cells. The loss of pigmentation was associated with the loss of endogenous tyrosinase expression at the activity and mRNA levels. In order to determine whether the p53 repression of tyrosinase mRNA involved modulation of tyrosinase promoter activity, transient transfection approaches involving p53 expression plasmid and construct containing chloramphenicol acetyl transferase (CAT) reporter gene linked to 270 bp tissue-specific tyrosinase promoter have been used. p53 specifically repressed CAT gene expression from the tyrosinase promoter and not from the Rous sarcoma virus promoter. These data suggest that in human melanoma p53 down-regulates the tissue-specific expression of tyrosinase gene and subsequent melanin synthesis.     

Ascidian neural crest-like cells: phylogenetic distribution, relationship to larval complexity, and pigment cell fate

Migratory neural crest-like cells, which express the cell surface antigen HNK-1 and develop into pigment cells, have recently been identified in the ascidian Ecteinascidia turbinata. Here we use HNK-1 expression as a marker to determine whether neural crest-like cells are responsible for pigment development in diverse ascidian species. We surveyed HNK-1 expression and tyrosinase activity in 12 ascidian species, including those with different adult organizations, developmental modes, and larval sizes and complexities. We observed HNK-1 positive cells in every species, although the timing of HNK-1 expression varied according to the extent of larval complexity. HNK-1 expression was initiated during the late tailbud stage in species in which adult features are formed precociously in large complex larvae. In contrast, HNK-1 positive cells did not appear until the swimming tadpole or juvenile stage in species with small simple larvae in which most adult features appear after metamorphosis. Double labeling experiments indicated that HNK-1 and tyrosinase are expressed in the same subset of pigment-forming mesenchymal cells in species with complex or simple larvae. In addition, the absence of HNK-1 and tyrosinase expression in albino morphs of the colonial ascidian Botryllus schlosseri suggested that the major fate of neural crest-like cells is to become pigment cells. The results suggest that ascidian neural crest-like cells and vertebrate neural crest cells had a common origin during chordate evolution and that their primitive function was to generate body pigmentation.     

Growth inhibition of murine melanoma by butyric acid and dimethylsulfoxide

Treatment of B16-F10 melanoma cells with dimethylsulfoxide (DMSO) or butyric acid (BA) inhibits cell growth and delays tumor appearance in syngeneic mice. Both agents induce morphological changes in these cells. Treatment of melanoma cells with DMSO results in a marked increase in tyrosinase activity and melanin content. BA, on the other hand, does not increase melanin content and decreases tyrosinase activity. The data show that there are marked differences in the effect of DMSO and BA on melanin biosynthesis, whereas both agents inhibit cell growth and cause a delay in tumor appearance. These findings indicate that decreased proliferation of melanoma cells and induction of melanin biosynthesis are not necessarily associated phenomena.     

Melanin accelerates the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH)

Although pigment melanin has long been though of as "inert," recent work has attested to its chemical reactivity. In this communication, we report that either commercial synthetic melanin prepared by persulfate oxidation of tyrosine ("Sigma melanin") or sepia melanin extracted from cuttlefish markedly accelerates the in vitro oxygenation of p-hydroxyanisole (MMEH), catalyzed by mushroom or B-16 melanoma tyrosinase. Kinetics of 4-methoxy-1,2-benzoquinone formation (lambda max = 413 nm) or of molecular O2 uptake were biphasic, with an initial slow rate ("lag time") followed by a fast linear increase. The biphasic response reflects an initial slow hydroxylation followed by a fast dehydrogenation. Added melanin markedly decreased the lag time but had little effect on subsequent dehydrogenation. Similar effects were observed for tyrosine itself. A complex between MMEH and melanin appears to be the "active" species in these reactions. The results indicate that melanin acts as an electron conduit, which accepts electrons from the substrate and transfers them to tyrosinase. The magnitude of the effect depends on the type of melanin as well as on its oxidation state. Kinetic analysis indicates that both melanins are very efficient at transferring electron to tyrosinase, and that Sigma melanin is roughly threefold more efficient than sepia melanin. The qualitative similarity of reaction between the synthetic and "natural" melanins suggests that the former may serve as a first approximation to the in vivo situation. On the other hand, the observed quantitative differences and the sensitivity of these results to the chemical state of melanin suggests that this methodology might eventually be adapted as a non-destructive probe of melanin in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation and Suppression of Melanogenesis in Mouse Melanoma Cells by Hybridization with Chick Embryonic Cells

Sendai virus-induced fusion of 6-thioguanine-resistant mouse melanoma cells (TG14) with various types of chick embryonic tissue cells resulted in the formation of hybrid cells. Isolated hybrid clones possessed almost complete sets of the cell chromosomes of the parent mouse and several dot-like chromosomes of the chick. Each type of hybrid clone showed characteristic tyrosinase activity that resulted in melanin production. An enhanced production of melanin was observed in the hybrids between not highly pigmented TG14 cells and retinal pigment cells. Electrophoretic analyses showed that banding patterns of tyrosinase were not of chick type but of mouse melanoma type. Numerous stage 111 and IV melanosomes of the mouse melanoma type were observed in pigmented hybrid clones. On the other hand, hybrid cells between mouse melanoma cells and chick embryonic liver cells exhibited lower tyrosinase activity.     

Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson's disease

Tyrosinase is a key enzyme in the synthesis of melanin in skin and hair and has also been proposed to contribute to the formation of neuromelanin (NM). The presence of NM, which is biochemically similar to melanin in peripheral tissues, identifies groups of neurons susceptible in Parkinson's disease (PD). Whether tyrosinase is beneficial or detrimental to neurons is unclear; whilst the enzyme activity of tyrosinase generates dopamine-quinones and other oxidizing compounds, NM may form a sink for such radical species. In the present study, we demonstrated that tyrosinase is expressed at low levels in the human brain. We found that mRNA, protein and enzyme activity are all present but at barely detectable levels. In cell culture systems, expression of tyrosinase increases neuronal susceptibility to oxidizing conditions, including dopamine itself. We related these in vitro observations to the human disease by assessing whether there was any genetic association between the gene encoding tyrosinase and idiopathic PD. We found neither genotypic or haplotypic association with three polymorphic markers of the gene. This argues against a strong genetic association between tyrosinase and PD, although the observed contribution to cellular toxicity suggests that a biochemical association is likely.     

Quantitative control of end products in the melanocyte lineage of the ascidian embryo.

Terminal amounts of tyrosinase (EC 1.10.3.1) activity and melanin pigment in the giant melanocytes of cleavage-arrestedCiona intestinalis (L.) embryos are regulated independently of cell size and number of nuclei in the cells. Embryos were cleavage-arrested in cytochalasin B at a time before the last two divisions of the melanocyte lineage took place. The resulting two giant melanocytes, one from each of the two bilateral melanocyte lineages, developed tyrosinase and melanin. The cells were about three times larger in volume than the normal larval melanocytes and each contained four nuclei instead of just one. Quantitative measurements of melanin synthesized and tyrosinase activity in embryos with the giant melanocytes revealed amounts identical to those found in normal embryos. This specification of exact quantities differs markedly from the situation in mammalian melanocytes where cell volume and gene dosage influence the extent of melanotic differentiation. Quantitative control of differentiation in ascidian melanocytes appears to be mediated by a cytoplasmic determinant segregated through the melanocyte lineage and inherited by one daughter at each division of the lineages.     

Pigment Deposition in Viscera Associated with Prolonged Chlorpromazine Therapy

Twelve physically healthy young adult mental hospital patients died unexpectedly while on prolonged chlorpromazine therapy. Five of them had clinically obvious pigmentation of the exposed skin. Two of these had impairment of vision as well. Autopsies were performed on all 12 patients. Extensive deposits of pigment (exhibiting the physical and histochemical properties of melanin) were present in macrophages in the dermis and throughout the reticuloendothelial system, and in the parenchymal cells of internal organs. The dopa tyrosinase reaction indicated increased melanocyte activity in the epidermis.The possible mechanism of production of this pigment is discussed, and the belief is expressed that the increased melanin production is due, at least partly, to the effect of chlorpromazine on the autonomic nervous system, blocking the production of pigment-lightening factors, of which melatonin is the most important. A short outline of contemplated further investigation is given.     

Effects of 4-tertiary butylphenol on the tyrosinase activity in human melanocytes.

Vitiligo is a common dermatological disorder characterized by the development of complete pigment loss from focal lesions that tends to increase in size over time. The etiology of vitiligo, resulting in the disappearance of functional melanocytes from involved skin, is not clearly understood. As a consequence, no satisfactory therapy has been developed. A subtype of vitiligo, termed 'occupational' or 'contact' vitiligo, is increased in individuals who are exposed to materials containing phenolic derivatives, such as 4-tertiary butylphenol (4-TBP). Phenolic derivatives are structurally similar to tyrosine, the initial substrate of tyrosinase in the biochemical synthesis of melanin. Therefore, it has been proposed that phenolic derivatives compete with tyrosine for hydroxylation by tyrosinase and interfere with the completion of melanin synthesis and/or generate cytotoxic intermediates. Our results demonstrated that 4-TBP competitively inhibited both tyrosine hydroxylase and dihydroxyphenylalanine (DOPA) oxidase activities of tyrosinase, i.e., the first two catalytic steps in the biochemical conversion of tyrosine to melanin in cultured human melanocytes. This inhibition occurred at concentrations that did not influence the viability of melanocytes. The tyrosinase activity inhibited by 4-TBP was recovered after removing the treatment. 4-TBP did not affect the function of other enzymes, such as succinate-tetrazolium reductase, acid phosphatase and sulfatase. Since depigmentation occurred without a cytotoxic response after exposure of melanocytes to low concentration of 4-TBP, it is unclear whether the interaction between 4-TBP and tyrosinase leads to the destruction of the melanocytes in 'contact/occupational' vitiligo.     

Mutational mapping of the catalytic activities of human tyrosinase.

Tyrosinase (EC 1.14.18.1) is a copper-containing metalloglycoprotein that catalyzes several steps in the melanin pigment biosynthetic pathway; the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (dopa) and the subsequent oxidation of dopa to dopaquinone. It has been proposed that tyrosinase is also able to oxidize 5,6-dihydroxyindole (DHI), a later product in the melanogenic pathway, to indole-5,6-quinone. Tyrosinase enzymatic activity is deficient in patients with classic type I oculocutaneous albinism (OCA), and more than 50 distinct mutations have now been identified in the tyrosinase genes of such patients. To determine the effects of the various tyrosinase gene mutations on the catalytic activities of the enzyme, we carried out site-directed mutagenesis of human tyrosinase cDNA, transiently expressed the mutant cDNAs in transfected HeLa cells, and assayed the resultant encoded proteins for tyrosine hydroxylase, dopa, and DHI oxidase activities, and resulting melanin production. The tyrosine hydroxylase activity of normal tyrosinase is thermostable, whereas its dopa oxidase and DHI oxidase activities are temperature-sensitive. Although all amino acid substitutions tested generally affected the dopa oxidase and DHI oxidase activities in parallel, several exerted distinctly different effects on the tyrosine hydroxylase activities. Together, these results confirm the DHI oxidase activity of mammalian tyrosinase and suggest that the dopa oxidase and DHI oxidase activities of tyrosinase share a common catalytic site, whereas the tyrosine hydroxylase catalytic site is at least partially distinct in the tyrosinase polypeptide.     

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.