Keratin Subunit Expression in Human Cultured Melanocytes and Mouse Neural Crest Cells Without Formation of Filamentous Structures

The synthesis of keratin is considered to occur in epithelial and epidermal cells. Previous studies have not reported on keratin synthesis within melanocytes that derive from neural crest cells. Epithelial and neural crest cells originally develop from ectodermal tissue. We previously reported that the expression of keratin is a universal phenomenon seen in cultured melanoma cell lines, as demonstrated by two-dimensional polyacrylamide gel electrophoresis, western blot, and electron microscopy analyses. To further investigate the specificity of keratin function in melanocytic cells, we first examined the presence of keratin proteins in cultured human melanocytes, and unexpectedly found keratin subunits in melanocytes by the above-mentioned procedures. The keratin (K) subunits were composed of K1, K5, K8, K10, K14, K16, and K18, together with vimentin. Neural crest cells, which contain immature embryonic melanocytes developing from ectoderm, already expressed keratins; however, under electron microscopy, the expressed keratin did not form filamentous structures. Although the ATP synthase α-chain, which is expressed universally in cultured epidermal tumor cell lines, was also expressed in cultured melanocytes and neural crest cells, a novel malignant melanoma-related protein (MMRP) was absent in melanocytes and neural crest cells. We concluded that keratin subunits are present in both cells, but do not construct keratin filaments

Effects of genic substitution at the pink-eyed dilution locus on the proliferation and differentiation of mouse epidermal melanocytes in vivo and in vitro

Cells positive to the dopa reaction (melanocytes) as well as to the combined dopa-premelanin reaction (melanoblasts and melanocytes) in the epidermis of C57BL/10JHir-p/p (pink-eyed dilution) mice were fewer and less reactive than in C57BL/10JHir (black, P/P) mice, suggesting that the proliferation and differentiation of p/p melanocytes are inhibited. To confirm the inhibitory effects of p gene on the proliferation and differentiation of epidermal melanocytes, we cultured epidermal cell suspensions of neonatal skins from P/P and p/p in a serum-free medium. The proliferation and differentiation of p/p melanoblasts/melanocytes in primary culture were greatly inhibited as compared to P/P melanoblasts/melanocytes. The morphology of p/p melanoblasts/melanocytes cultured in melanocyte growth medium, though non-pigmented, was similar to P/P melanocytes; namely, dendritic, polygonal, or epithelioid. About 8% of p/p cells cultured in melanocyte growth medium were positive to the dopa reaction, and about 25% were reactive to the combined dopa-premelanin reaction. Eumelanin content in p/p was extremely reduced compared to P/P. The immunocytochemical staining of p/p melanoblasts/melanocytes revealed that they are negative to tyrosinase, but reactive to tyrosinase-related protein (TRP)-1, TRP-2, and c-kit. However, the reactivities in p/p were lower than in P/P . Although the differentiation of p/p melanoblasts was not induced by endothelin (ET)-1, ET-2, and ET-3, the proliferation of p/p melanoblasts was stimulated by them. These results suggest for the first time that p gene exerts its influence on the proliferative activities of mouse epidermal melanoblasts by affecting the regulatory mechanisms dependent on the function of ETs

Excess Tyrosine rescues the reduced activity of the proliferation and differentiation of cultured recessive yellow melanocytes of neonatal mouse epidermis

The mouse recessive yellow (Mc1re) is a loss-of-function mutation in the receptor for arpha-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 Mc1re 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 Mc1re-locus, black melanocytes. The differentiation of melanocytes was also delayed and inhibited in recessive yellow mice. Tyrosinase (Tyr) activity and Tyr-related protein 1 (Tyrp1) and Tyrp2 (dopachrome tautomerase, Dct) expressions were decreased and, in addition, the maturation of stage IV melanosomes was inhibited. Excess L-tyrosine added to the culture media rescued the reduced activity of proliferation of melanocytes. L-tyrosine also stimulated Tyr activity and Tyrp1 and Tyrp2 expressions as well as the maturation of stage IV melanosomes and pigmentation. These results suggest that the Mc1re mutation affects the proliferation and differentiation of melanocytes and L-tyrosine rescues the reduced proliferative and differentiative activities by stimulating Tyr activity and Tyrp1 and Tyrp2 expressions as well as melanosome maturation.14th Meeting of the European Society for Pigment Cell Researc

The slaty mutation affects eumelanin and pheomelanin synthesis in mouse melanocytes

The slaty (slt) mutation is known to reduce the activity of dopachrome tautomerase (Dct)/tyrosinase (Tyr)-related protein (TRP)-2 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 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. Tyr activity detected by dopa reaction as well as staining of Dct/TRP-2 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 gene 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 activities of Dct/TRP-2 and Tyr