Excess Tyrosine Stimulates Eumelanin and Pheomelanin Synthesis in Cultured Slaty Melanocytes from Neonatal Mouse Epidermis (original) (raw)
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The slaty mutation affects eumelanin and pheomelanin synthesis in mouse melanocytes
European Journal of Cell Biology, 2006
The slaty (Dct slt ) mutation is known to reduce the activity of dopachrome tautomerase (DCT) in melanocytes. However, it is unknown whether the reduced DCT activity leads to a defect in the proliferation and differentiation of mouse melanocytes. To address this point, the proliferation and differentiation of neonatal melanocytes from Dct slt / Dct slt congenic mice in serum-free primary culture were investigated in detail. The proliferation of slaty epidermal melanoblasts/melanocytes in culture did not differ from that of wild-type mice. However, the differentiation was greatly inhibited. Tyrosinase (TYR) activity detected by dopa reaction as well as staining of DCT in slaty melanocytes was greatly reduced. The content of eumelanin in cultured slaty melanocytes was reduced, whereas the content of pheomelanin in media derived from cultured 7.5-day-old slaty melanocytes was greatly increased. The contents of eumelanin and pheomelanin in the neonatal slaty epidermis and dermis were reduced, except that the pheomelanin content in 3.5-day-old dermis was increased. These results suggest that the slaty mutation affects both eumelanin and pheomelanin synthesis in developmental stage-specific and skin site-specific manners, and, in addition, the gene controls the differentiation of melanocytes via the regulation of activity of TYR in addition to its own DCT.
Journal of Cellular Physiology, 2002
The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mouse at the pink-eyed dilution (p) locus (black, C57BL/10JHir-P/P) and their congenic mutant mouse (pink-eyed dilution, C57BL/10JHir-p/p) were cultured with a serum-free melanocyte growth medium supplemented with additional L-tyrosine (Tyr) from initiation of the primary culture. L-Tyr inhibited the proliferation of P/P melanocytes in a dose-dependent manner, whereas L-Tyr stimulated the proliferation of p/p melanoblasts and melanocytes regardless of dose. On the other hand, L-Tyr stimulated (P/P) or induced (p/p) the differentiation of epidermal melanocytes in a dose-dependent manner. In both P/P and p/p melanoblasts and melanocytes cultured with 2.0 mM L-Tyr for 14 days, slight increases in contents of eumelanin marker, pyrrole-2,3,5-tricarboxylic acid (PTCA) and pheomelanin marker, aminohydroxyphenylalanine (AHP) were observed. The average number of total melanosomes (stages I, II, III, and IV) per P/P melanocyte was not changed by L-Tyr treatment, but the proportion of stage IV melanosomes in the total melanosomes was increased. On the contrary, in p/p melanoblasts and melanocytes L-Tyr increased dramatically the number of stage II, III, and IV melanosomes as well as the proportion of stage III melanosomes. Contents of PTCA and eumelanin precursor, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) of cultured media in p/p melanocytes were much more greatly increased than in P/P melanocytes. However, contents of AHP and pheomelanin precursor, 5-Scysteinyldopa (5-S-CD) of cultured media in p/p melanocytes were increased in a similar tendency to P/P melanocytes. These results suggest that p/p melanocytes in the primary culture are induced to synthesize eumelanin by excess L-Tyr, but difficult to accumulate them in melanosomes.
Journal of Investigative Dermatology, 2004
The quality, quantity and distribution of melanosomes in epidermis play a crucial role in the determination of skin color and its sensitivity to UV radiation. Melanocyte cultures originating from individuals with light and dark skin types were grown in media with varying concentration of L-tyrosine. Melanosomal melanin content and the size of the organelles were measured after subcellular fractionation. In light-skin type cells, increased melanin production resulted in a more elliptical shape of melanosomes. In melanosomes that constitutively produce more melanin, the tyrosine-induced melanogenesis caused enlargement in all dimensions. X-ray microanalysis provided evidence that the increase in sulfur content induced by high tyrosine concentration was more prominent in the melanosomes from light skin types. A ratio between pheomelanin and eumelanin found in light-skin type melanosomes by HPLC was increased more markedly than that in melanosomes from dark skin melanocytes. These findings suggest that the melanocytes of lightskinned individuals exhibit a preference for pheomelanogenesis. Pheomelanin production is a thiol-consuming process and that might increase the risk of oxidation stress in these cells. This fact, together with the limited ability of pheomelanin to absorb UV radiation may lead to an elevated skin cancer risk among light-skinned individuals.
European Journal of Cell Biology, 2007
The murine recessive yellow (Mc1r e ) is a loss-of-function mutation in the receptor for a-melanocyte-stimulating hormone, melanocortin receptor 1 (Mc1r) and produces yellow coats by inducing pheomelanin synthesis in hair follicular melanocytes. However, it is not known whether the Mc1r e mutation affects the proliferation and differentiation of melanocytes. In this study, the proliferation and differentiation of recessive yellow epidermal melanocytes cultured in dibutyryl cyclic AMP-supplemented serum-free medium were investigated in detail. The melanocytes produced mainly eumelanin in this culture system. The proliferation of recessive yellow melanocytes was decreased compared with that of wild-type at the e-locus, black melanocytes. The differentiation of melanocytes was also delayed and inhibited in recessive yellow mice. Tyrosinase (TYR) activity and TYR-related protein 1 (TRP1) and TRP2 (dopachrome tautomerase, DCT) expressions were decreased and, in addition, the maturation of stage IV melanosomes was inhibited. Excess L-tyrosine (L-Tyr) added to the culture media rescued the reduced activity of proliferation of melanocytes. L-Tyr also stimulated TYR activity and TRP1 and TRP2 expressions as well as the maturation of stage IV melanosomes and pigmentation. These results suggest that the Mc1r e mutation affects the proliferation and differentiation of melanocytes and L-Tyr rescues the reduced proliferative and differentiative activities by stimulating TYR activity and TRP1 and TRP2 expressions as well as melanosome maturation.
Variations in melanin formation by cultured melanocytes from different skin types
Archives of Dermatological Research, 1998
In many laboratories, culturing skin melanocytes has become a routine research activity. However, recent investigations have revealed that the quality and quantity of the pigment formed in the cultured cells may differ significantly from those of the original skin pigment cells. To shed more light on this issue, we examined the influence of different culture media on pigment production. We showed that there were notable passage-to-passage variations in the synthesis of melanin. This was particularly true for phaeomelanin. It is therefore advisable to analyse the melanin in the cells before the start of experiments. In spite of the variations, basic differences in the pigmentation pattern between melanocytes isolated from light-skinned and dark-skinned individuals remained preserved in the corresponding cultures as observed by electron microscopy. Also, the total melanin content was higher in a skin type VI melanocyte culture than in skin type I and II melanocyte cultures. In contrast to total melanin, the phaeomelanin concentration of skin type VI cells was similar to that of the skin type I melanocytes. With higher L-tyrosine concentrations in the medium, as well as increased eumelanin synthesis, phaeomelanogenesis was also stimulated in all cultures tested. This stimulation was particularly prominent in skin type I melanocytes.
Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society, 2003
Changes in the proliferation and differentiation of epidermal melanocytes derived from newborn mice wild-type at the pink-eyed dilution (p) locus (P/P) and from congenic mice mutant at that locus (p/p) were investigated in serum-free primary culture, with or without the addition of L-Tyr. Incubation with added L-Tyr inhibited the proliferation of P/P melanocytes in a concentration-dependent manner and inhibition was gradually augmented as the donor mice aged. In contrast, L-Tyr stimulated the proliferation of p/p melanoblasts-melanocytes derived from 0.5-day-old mice, but inhibited their proliferation when derived from 3.5- or 7.5-day-old mice. L-Tyr stimulated the differentiation of P/P melanocytes. However, almost all cells were undifferentiated melanoblasts in control cultures derived from 0.5-, 3.5- and 7.5-day-old p/p mice, but L-Tyr induced their differentiation as the age of the donor mice advanced. The content of the eumelanin marker, pyrrole-2,3,5-tricarboxylic acid as well...
European Journal of Cell Biology, 1998
To examine the effects of coat-color genes on the proliferation and dif. ferentiation of mouse epidermal melanocytes, we cultnred epidermal · cell suspensions derived from neonatal skins of C57BU10Jifu (black) and its congenic mice carrying agouti, brown, albino, dilute, and pink· eyed dilution genes in a serum-free medium supplemented with dibutyryl adenosine 3' ,5'-cyclic monophosphate. The proliferative rates of agouti, brown and dilute black melanocytes were similar to that of black melanocytes, while those of albino and pink-eyed black melanocytes were about one-half of that of black melanocytes. The morphology of albino and pink-eyed black melanocytes, though nonpigmented, was similar to black melanocytes; namely, dendritic, polygonal or epithelioid. Dilute black melanocytes also possessed the similar morphology, whereas their melanosomes were accumulated in the perinuclear region. Dopa-melanin depositions after dopa reaction in brown and dilute black melanocytes were greater than in black and agouti melanocytes. Although dopa-melanin depositions were not observed in albino melanocytes, about 8 % of pink-eyed black melanocytes were positive to dopa reaction~ Silver depositions after combined dopa-premelanin reaction in agouti, brown and dilute black melanocytes were similar to that in black melanocytes. Although albino melanocytes were devoid of silver depositions, about 25 % of pink-eyed black melanocytes were positive to the reaction. Pyrrole-2,3,5tricarboxylic acid (PTCA, degradation product of eumelanin) contents in agouti and dilute black melanocytes were slighdy lower than in black melanocytes, while that in brown melanocytes was reduced to one-third. In contrast, PTCA contents in albino and pink-eyed black melanocytes were reduced to less than 0.5 %. Aminohydroxyphenylalanine (AHP, degradation product of pheomelanin) contents did not differ among these melanocytes. These results suggest that the coatcolor genes exert their inftuences on the proliferation and differentiation of mouse epidermal melanocytes by affecting tyrosinase activity, melanosome maturation and transport, and eumelanin synthesis.
Chemical Analysis of Melanins and its Application to the Study of the Regulation of Melanogenesis
Pigment Cell Research, 2000
show that tyrosinase activity is the most important factor that regulates the tractable chemical properties, the heterogeneity in their switch of melanogenesis, with lower tyrosinase activities fa-structural features, and the lack of methods to split melanin polymers into monomer units. To overcome this difficulty, voring pheomelanogenesis; further suppression of melanogenwe developed a rapid and sensitive method for quantitatively esis results in a lack of pigment production. 3) In cultured analyzing eumelanin and pheomelanin in biological samples melanocytes, the concentrations of tyrosine and cysteine, that is based on the formation of pyrrole-2,3,5-tricarboxylic and their ratio in the medium, are important in determining the concentrations of eumelanin and pheomelanin produced acid and/or aminohydroxyphenylalanine followed by HPLC and their ratio in the cells. In conclusion, our HPLC micro-determination. The method has been applied to the study of melanogenesis. The results summarized in this review are: 1) analytical method for characterizing eumelanin and pheomelanin has become a useful tool for the study of melano-Biochemical studies show that in the process of mixed genesis. melanogenesis, cysteinyldopas are produced first, which are then oxidized to give pheomelanin; following cysteine depletion, eumelanin is then deposited on the preformed pheome-
Melanins and melanogenesis: methods, standard, protocols
2013
Despite considerable advances in the past decade, melanin research still suffers from the lack of universally accepted and shared nomenclature, methodologies and structural models. This paper stems from the joint efforts of chemists, biochemists, physicists, biologists and physicians with recognized and consolidated expertise in the field of melanins and melanogenesis, who critically reviewed and experimentally revisited methods, standards and protocols to provide for the first time a consensus set of recommended procedures to be adopted and shared by researchers involved in pigment cell research. Aim of the paper is the definition of an unprecedented frame of reference built on cuttingedge knowledge and state-of-the-art methodology, to enable reliable comparison of results among laboratories and new progress in the field based on standardized methods and shared information.
The Mouse Brown ( b/Tyrp1 b ) Allele Does Not Affect Pheomelanin Synthesis in Mice
Zoological Science, 2014
B (Tyrp1 + ), the wild type allele at the mouse brown locus, produces black eumelanin, while b (Tyrp1 b ), the recessive allele, produces brown eumelanin and exhibits lower tyrosinase (Tyr)-related protein 1 (Tyrp1) activity. However, it is unknown whether melanocyte proliferation and differentiation are affected by the Tyrp1 b mutation. The proliferation rate of brown (C57BL/10JHir (B10)-Tyrp1 b / Tyrp1 b ) melanocytes cultured in a serum-free melanocyte proliferation medium (MDMD) was similar to that of black (B10-Tyrp1 + /Tyrp1 + ) melanocytes. Although brown melanocytes exhibited normal morphology, their pigmentation was lower than that of black melanocytes. However, Tyr activity in brown melanocytes was increased both in vivo and in vitro.