Pheomelanin Synthesis Research Articles

Agouti Signaling Protein Inhibits Melanogenesis and the Response of Human Melanocytes to α-Melanotropin

In mouse follicular melanocytes, the switch between eumelanin and pheomelanin synthesis is regulated by the extension locus, which encodes the melanocortin-1 receptor (MC1R) and the agouti locus, which encodes a novel paracrine-signaling molecule that inhibits binding of melanocortins to the MC1R. Human melanocytes express the MC1R and respond to melanotropins with increased proliferation and eumelanogenesis, but a potential role for the human homolog of agouti-signaling protein, ASIP, in human pigmentation has not been investigated. Here we report that ASIP blocked the binding of alpha-melanocyte-stimulating hormone (alpha-MSH) to the MC1R and inhibited the effects of alpha-MSH on human melanocytes. Treatment of human melanocytes with 1 nM-10 nM recombinant mouse or human ASIP blocked the stimulatory effects of alpha-MSH on cAMP accumulation, tyrosinase activity, and cell proliferation. In the absence of exogenous alpha-MSH, ASIP inhibited basal levels of tyrosinase activity and cell proliferation and reduced the level of immunoreactive tyrosinase-related protein-1 (TRP-1) without significantly altering the level of immunoreactive tyrosinase. In addition, ASIP blocked the stimulatory effects of forskolin or dibutyryl cAMP, agents that act downstream from the MC1R, on tyrosinase activity and cell proliferation. These results demonstrate that the functional relationship between the agouti and MC1R gene products is similar in mice and humans and suggest a potential physiologic role for ASIP in regulation of human pigmentation.

Interactions of α-Melanotropin and Agouti on B16 Melanoma Cells: Evidence for Inverse Agonism of Agouti

α-Melanocyte-stimulating hormone (α-MSH, α-melanotropin) and agouti control the switch between eumelanin and pheomelanin synthesis in mammalian melanocytes. Here we investigated interactions between α-MSH, agouti protein, cAMP elevating agents and phorbol ester on mouse B16 melanoma cells. Agouti (Kd 3.7nmol/l) and α-MSH (Kd 2.3 nmol/l) had similar affinities to the MC1 melanocortin receptor. Both α-MSH and agouti induced MC1 receptor down-regulation. Agouti antagonized melanogenesis induced by α-MSH, forskolin, cholera toxin (CT), and pertussis toxin (PT). It also reduced the constitutive melanin formation of long-term cultures. Cell proliferation was inhibited by agouti (43% at 100 nM). This effect was reversed by α-MSH, forskolin, or CT. B16-G4F cells, a cell variant that lacks the MC1 receptor, did not respond to agouti. From these results we conclude that agouti shows the characteristics of an inverse agonist acting through the MC1 receptor.

Characterization of melanogenesis in normal human epidermal melanocytes by chemical and ultrastructural analysis.

Chemical and ultrastructural studies were conducted to define the relationship between type of melanogenesis and fine structures of melanosomes in normal human epidermal melanocytes. Chemical analysis of epidermal melanin demonstrated that the ratio of eumelanin/pheomelanin varied individually, ranging from 1.31 to exclusively eumelanic. Ultrastructural analysis of fine structures of melanosomes revealed that spheroid melanosomes were frequently observed in melanocytes of the epidermis whose eumelanin/ pheomelanin ratio was less than 5. Conversely, ellipsoid melanosomes predominated in melanocytes of the epidermis whose ratio was more than 10. On the basis of these findings, it seems reasonable to conclude that 1) normal human epidermal melanocytes synthesize both eumelanin and pheomelanin and 2) pheomelanin synthesis may be characterized by the presence of spheroid melanosomes whereas eumelanin synthesis is ascribed to ellipsoid melanosomes.

The interaction of agouti signal protein and melanocyte stimulating hormone to regulate melanin formation in mammals.

Important regulatory controls of melanogenesis that operate at the subcellular level to modulate the structural and/or the functional nature of the melanins and melanin granules produced in melanocytes are reviewed. Melanocyte stimulating hormone and agouti signal protein have antagonistic roles and possibly opposing mechanisms of action in the melanocyte. In the mouse, melanocyte stimulating hormone promotes melanogenic enzyme function and elicits increases in the amount of eumelanins produced, while agouti signal protein reduces total melanin production and elicits the synthesis of pheomelanin rather than eumelanin. We are now beginning to understand the complex controls involved in regulating this switch at the molecular and biochemical levels. The quality and quantity of melanins produced by melanocytes have important physiological consequences for melanocyte function and undoubtedly play important roles in the various functions of the melanins per se, including hair and skin coloration and photoprotection.

An Ultrastructural Study of Melanocytes and Melanosomes in the Skin and Hair Bulbs of Rufous Albinos

We have examined hair bulb and skin melanocytes of rufous albinos from Southern Africa to further characterize this form of albinism. In the skin melanocytes we find both eumelanosomes and pheomelanosomes at various stages of melanization and, in addition, there appeared to be many aberrant incompletely melanized melanosomes. On average, rufous melanosomes are 30% smaller than normal black skin melanosomes. In the keratinocytes, the melanosomes are packaged into distinct aggregations, whereas in normal black skin, they occur singly. We suggest that the reddish skin color of these albinos is a consequence of an increase in the pheomelanin synthesis resulting in a raised pheomelanin/eumelanin ratio and that the aggregation of melanosomes results in a skin color slightly lighter than normal. In hair bulb melanocytes, only eumelanosomes were seen and these were mostly incompletely melanized. These findings correlate with our visual observations that the hair color of Southern African albinos is very pale (light brown or ginger). Based on our observations, we speculate on the possible cause of rufous albinism.

Melanin pigments and melanosomal proteins as differentiation markers unique to normal and neoplastic melanocytes.

This report introduces some aspects of our current basic research focus on the unique metabolic pathways within the melanocyte. Using this approach, we hope to gain a better understanding of the pathophysiology of malignant melanoma and develop early laboratory diagnostic tests for this disease. Specifically, we will discuss that: 1) the synthesis of pheomelanin is markedly increased in malignant melanoma and dysplastic melanocytic nevi; 2) high levels of metabolites of pheomelanin and eumelanin can be detected in the urine and blood of patients with metastatic melanoma; 3) this release of melanin metabolites appears to correlate with tumor thickness and tumor load, including the extent of metastasis; 4) the synthesis of melanosomal proteins also becomes aberrant in malignant melanoma; and 5) this abnormal melanosome synthesis can be utilized in the identification of antigenic epitopes that are uniquely expressed in malignant melanoma. We believe that this synthesis and secretion of abnormal melanin pigment and melanosomal proteins (human melanosome-specific antigen) would be useful for the development of early laboratory diagnostic and monitoring tools for malignant melanoma. In addition, we also report the detection of pheomelanin component in "normal" unexposed skin; however, the relative amount of pheomelanin in the skin does not reflect hair color (e.g., red hair). The nature of this pheomelanin component in the skin needs to be further clarified.

Melanin Pigments and Melanosomal Proteins as Differentiation Markers Unique to Normal and Neoplastic Melanocytes

This report introduces some aspects of our current basic research focus on the unique metabolic pathways within the melanocyte. Using this approach, we hope to gain a better understanding of the pathophysiology of malignant melanoma and develop early laboratory diagnostic tests for this disease. Specifically, we will discuss that: 1) the synthesis of pheomelanin is markedly increased in malignant melanoma and dysplastic melanocytic nevi; 2) high levels of metabolites of pheomelanin and eumelanin can be detected in the urine and blood of patients with metastatic melanoma; 3) this release of melanin metabolites appears to correlate with tumor thickness and tumor load, including the extent of metastasis; 4) the synthesis of melanosomal proteins also becomes aberrant in malignant melanoma; and 5) this abnormal melanosome synthesis can be utilized in the identification of antigenic epitopes that are uniquely expressed in malignant melanoma. We believe that this synthesis and secretion of abnormal melanin pigment and melanosomal proteins (human melanosome-specific antigen) would be useful for the development of early laboratory diagnostic and monitoring tools for malignant melanoma. In addition, we also report the detection of pheomelanin component in "normal" unexposed skin; however, the relative amount of pheomelanin in the skin does not reflect hair color (e.g., red hair). The nature of this pheomelanin component in the skin needs to be further clarified.

Tyrosinase and the regulation of coat color changes in C3H-HeAvy mice.

Hair follicular tyrosinase activity was measured during hair growth in neonatal, pubertal, and adult C3H-HeAvy mice that show differences in coat color as a result of changes in the synthesis of eumelanin and pheomelanin. Tyrosinase activity increased during hair growth in all mice but higher levels were found at puberty, when the mice grow a dark, eumelanin coat of hair, than during early and adult life, when the hair follicular melanocytes produce mainly pheomelanin. This suggests that tyrosinase is more important for the synthesis of eumelanin than that of pheomelanin. The increased tyrosinase activity associated with eumelanogenesis in the pubertal mice could not be explained by enhanced transcription of the tyrosinase gene or enzyme synthesis and appeared to be the result of a post-translational activation. Such an activation of tyrosinase was lacking in the neonatal and adult mice; in the latter this was accompanied by a reduction in the glycosylation of tyrosinase and the proportion of enzyme associated with the melanosomal fraction. Our findings suggest that post-translational mechanisms are important control points in the regulation of tyrosinase and that differences in their level of activation are responsible for determining the patterns of melanogenesis in the C3H-HeAvy mice, but it is still not clear how these mechanisms are regulated. Although cyclic AMP increased tyrosinase synthesis it had no post-translation activating effect. The neonatal mice, unlike their pubertal and adult counterparts, also lacked dopachrome converting activity and TRP tyrosinase-related protein-1 expression.(ABSTRACT TRUNCATED AT 250 WORDS)

After Dopachrome?

Dopachrome, an intermediate in melanin biosynthesis, exhibits some unusual properties. At physiologic pH (e.g., pH 6-8) it is unstable and spontaneously loses its carboxyl group to form 5,6-dihydroxyindole (DHI) and CO2. However, over this same pH range, if various metals or a melanocyte-specific enzyme are present, it rapidly rearranges to its isomer form--5,6-dihydroxyindole-2-carboxylic acid (DHICA)--which is far more stable than dopachrome in its ability to retain the carboxyl group. Whether or not the carboxyl group is retained could have important implications for the regulation of melanogenesis, since in the presence of oxygen DHI spontaneously forms a black precipitate, whereas DHICA forms a golden-brown solution. The solubility of "DHICA-melanin" is due to the presence of carboxyl groups, which provide negative charges and hydrophilicity. Thus, in vivo, the extent to which dopachrome is converted to DHI or DHICA may well influence the solubility and color of the melanin formed. The purpose of this article is to review recent findings in these areas and to discuss the possible significance of dopachrome conversion in the regulation of melanogenesis and color formation.

Incorporation of sulfhydryl compounds into melanins in vitro

Cysteine is known to be involved in pheomelanin synthesis. Available evidence, however, indicates that glutathione may also play an important role in melanogenesis. This in vitro study clarifies how cysteine and glutathione participate in melanogenesis. Melanins were prepared by tyrosinase oxidation of 3,4-dihydroxyphenylalanine (dopa) with various amountsof cysteine or glutathione, and were subjected to HCl hydrolysis. Synthetic melanins and the hydrolyzed melanins gave N/S molar ratios corresponding to those calculated from the ratio of dopa to cysteine or glutathione. On hydriodic acid hydrolysis, dopa plus cysteine-melanins and dopa plus glutathione-melanins gave aminohydroxyphenylalanine and cysteine, respectively, the yields of which were proportional to the sulfur content. The results indicate that cysteine is integrated into benzothiazine units (pheomelanins) while glutathione is connected to dihydroxyindole units (eumelanins)_ with the retention of glutathione moiety. Analysis of natural melanins, prepared by HCl hydrolysis of Sepia, B16, and Harding-Passey melanosomes, indicates that sulfur (0.4–1.4%) in these natural melanins may be derived artificially from the reaction of melanins with cysteine or cystine in the course of HCl hydrolysis.

Effect of DOPA-Loading on Glutathione-dependent 5-S-Cysteinyldopa Genesis in Melanoma Cells in Vitro

The effect of dopa, cysteine, and glutathione on 5-S-cysteinyldopa (5-S-CD) genesis in melanoma cells cultured in normal and tyrosine- and cysteine-free media has been studied. In normal media only melanotic melanoma cells have been found to secrete 5-S-CD into the medium. In the presence of dopa and cysteine, both, media incubated with and without cells have been found to produce 5-S-CD. In the presence of dopa and glutathione, however, cell-free media did not show the presence of 5-S-CD. In contrast melanoma cell-cultured media has been found to contain large quantities of this amino acid. The optimum condition for maximum production of 5-S-CD via glutathione-dependent pathway has been found to be at the dopa concentration of 10(-5) M when glutathione is present at the concentration of 10(-5) M in the culture medium. Thus dopa concentration with regards to glutathione is 1:1 on the molar basis which is twice the dopa concentration required in in vitro noncellular tyrosinase system. It is suggested that higher dopa requirement in our melanoma cell culture system reflects the co-existence of eu- and pheomelanin synthesis taking place according to their genetically predetermined proportions.

Dermal-epidermal interactions and the action of alleles at the agouti locus in the mouse

In the mouse, alleles at the agouti locus determine eumelanin or pheomelanin synthesis by the follicular melanocytes. Previous studies have identified the dermis as the site of action of these alleles. However, a recent investigation utilizing the yellow ( A y ) allele suggested a possible role of the epidermis in the expression of agouti locus alleles. Using dermal-epidermal recombinations of embryonic skin of various agouti genotypes, the present investigation supports the role of both the dermis and epidermis. If nonagouti ( a a ) dermis is recombined with agouti ( A A ) epidermis, the resulting hairs are pigmented in the nonagouti pattern. The reciprocal recombination of agouti dermis and nonagouti epidermis results in hairs pigmented in the agouti pattern. The recombinations of yellow ( A y a ) dermis and agouti or extreme nonagouti ( a e a e ) epidermis result in hairs completely pigmented in the yellow pattern (pheomelanin). However, when extreme nonagouti or agouti dermis is recombined with yellow epidermis, the resulting hairs are completely pigmented with pheomelanin. Similar results occur in recombinations of “young” yellow epidermis (13 days) and “old” dermis (17 days) even though dermal papillae are present. The role of dermal-epidermal interactions in the expression of agouti alleles as well as possible explanations for the unique action of the yellow allele are discussed.

Biosynthesis of a Thiouracil Pheomelanin in Embryonic Pigment Cells Exposed to Thiouracil

Abstract [2-14C]Thiouracil and [35S]thiouracil are incorporated into the melanin of chick retinal pigment epithelium incubated in vitro. This synthesis of a thiouracil pheomelanin (thiol-containing melanin) can be used to measure the rate of melanin synthesis in living cells. Thiouracil incorporation is dependent on tyrosinase activity: incorporation is inhibited by phenylthiourea, a tyrosinase inhibitor, and there is a proportional relationship between thiouracil incorporation and the level of tyrosinase activity in pigment tissues. Other chick embryo tissues do not incorporate thiouracil, but small quantities are incorporated by embryo tissues containing tyrosine hydroxylase. Radioactivity is retained in melanin prepared from isolated melanosomes of pigment tissues incubated in [2-14C]thiouracil. A comparison of [35S]thiouracil and l-[2-14C]tyrosine incorporations into melanin indicates that 1.5 moles of thiouracil are incorporated for each mole of tyrosine. These results are consistent with the hypothesis that thiouracil condenses with the quinone intermediates in the melanin pathway and is incorporated into the melanin polymer as part of the subunit structure.

The ultrastructural effects of the I and S loci upon black-red melanin differentiation in the fowl

The dominant white and silver mutants of the fowl were examined ultrastructurally and histochemically because of their unique effects upon black-red melanin differentiation—dominant white bleaches eumelanin, silver whitens pheomelanin. Both eumelanin- and pheomelanin-producing melanocytes were studied in birds of the dominant white, silver, and standard genotypes. Eumelanocytes and pheomelanocytes of all three genotypes possessed tyrosinase as revealed by DOPA incubation tests. This shows that the white areas of the two mutants are not due to the absence of a competent pigment cell system. Standard and silver eumelanocytes were melanogenically very active, exhibiting a high concentration of melanosomes. Dominant white eumelanocytes, however, possessed only a threshold number (one-half standard) of irregularly shaped, partially melanized melanosomes. Pheomelanocytes of all three genotypes were melanogenically quiescent during early development containing only a threshold number (one-half eumelanocyte) of unmelanized premelanosomes. Eumelanocytes of all three genotypes possessed dendrites that were capable of transferring melanosomes into keratinocytes. Dominant white melanocytes transferred fewer because less melanosomes were available. Transferred pheomelanosomes of the standard and dominant white hackles were solidly melanized and frequently of a compound nature. Silver pheomelanosomes, however, were only slightly melanized although compound premelanosomes were frequently observed. Standard pheomelanocytes were sulfhydryl positive, and silver pheomelanocytes were sulfhydryl negative as revealed by histochemical tests. This relates sulfhydryl groups to pheomelanin deposition. Based on the results, a model of melanogenesis is proposed which suggests that three differences exist between eumelanin formation and pheomelanin synthesis.

Origin of Pigment Cells from the Neural Crest in the Mouse Embryo

testing the pigment-forming potency of various portions of mouse embryos of a potentially pigmented (black) strain, evidence was obtained which suggested that the migratory pigment-forming cells, the melanophores, take their origin from the neural crest, as in other vertebrates, e.g., amphibians and birds.The results of grafting a variety of embryonic mouse tissues, such as skin ectoderm plus the underlying mesoderm, somites with and without the adjacent neural tube, limb buds and prospective limb buds, from various body levels, showed