Effects of melanogenesis-inducing nitric oxide and histamine on the production of eumelanin and pheomelanin in cultured human melanocytes

Melanin pigments produced in human melanocytes are classified into two categories; black coloured eumelanin and reddish-yellow pheomelanin. Stimulation of melanocytes with alpha-melanocyte-stimulating hormone (alpha-MSH), one of several melanogenic factors, has been reported to enhance eumelanogenesis to a greater degree than pheomelanogenesis, which contributes to hyperpigmentation in skin. Nitric oxide (NO) and histamine are also melanogenesis-stimulating factors that are released from cells surrounding melanocytes following ultraviolet (UV) irradiation. In this study, the effects of NO and histamine on the ratio of eumelanin and pheomelanin were examined in human melanocytes, and then compared with that of alpha-MSH. The amounts of eumelanin and pheomelanin were quantified using high-performance liquid chromatography analysis after oxidation and hydrolysis of melanin. Melanogenesis was induced by the addition of alpha-MSH, NO, or histamine to melanocytes. The amount of eumelanin production significantly increased with independent stimulation by these melanogenic factors, especially histamine, while that of pheomelanin significantly increased with alpha-MSH and NO, but only slightly with histamine. As a result, the ratio of eumelanin and pheomelanin increased significantly with the addition of NO or histamine. These results suggest that NO and histamine, as in the case of alpha-MSH, may contribute to UV-induced hyperpigmentation by enhancing eumelanogenesis.     

Possible involvement of ERK 1/2 in UVA-induced melanogenesis in cultured normal human epidermal melanocytes

UV-induced melanogenesis is a well known physiological response of human skin exposed to solar radiation; however, the signaling molecules involved in the stimulation of melanogenesis in melanocytes following UV exposure remain unclear. In this study we induced melanogenesis in vitro in normal human epidermal melanocytes using a single irradiation with UVA at 1 kJ/m2 and examined the potential involvement of mitogen-activated protein kinases (MAPK) as UVA-responsive signaling molecules in those cells. UVA irradiation did not affect the proliferation of melanocytes, but it did increase tyrosinase mRNA expression, which reached a maximum level 4 hr after UVA irradiation. The amount of tyrosinase protein, as quantitated by immunoblotting, was also increased at 24 hr following UVA irradiation. Among the MAPK examined, extracellular signal-related kinase (ERK) 1/2 was phosphorylated within 15 min of UVA irradiation, but no such phosphorylation was observed for c-Jun N-terminal kinases (JNK) or p38. Accordingly, the activity of ERK1/2 was also increased shortly after UVA irradiation. These responses of ERK1/2 to UVA irradiation were markedly inhibited when cells were pre-treated with N-acetyl-L-cysteine, an antioxidant, or with suramin, a tyrosine kinase receptor inhibitor. The formation of (6-4)photoproducts or cyclobutane pyrimidine dimers was not detected in cellular DNA after UVA irradiation. These findings suggest that a single UVA irradiation-induced melanogenesis is associated with the activation of ERK1/2 by upstream signals that originate from reactive oxygen species or from activated tyrosine kinase receptors, but not from damaged DNA.     

Excess tyrosine stimulates eumelanin and pheomelanin synthesis in cultured slaty melanocytes from neonatal mouse epidermis

The mouse slaty (Dct(slt)) mutation is known to reduce the activity of dopachrome tautomerase (DCT). The reduced DCT activity inhibits melanosome maturation and reduces the melanin content in the skin, hair and eyes. It is not known whether eumelanin and pheomelanin synthesis in slaty melanocytes is modulated by melanogenic factors. In this study, to address this point, epidermal melanocytes derived from 0.5-, 3.5- and 7.5-day-old wild-type mice (Dct(+)/Dct(+) at the slaty locus) and from congenic mice mutant (Dct(slt) /Dct(slt) at that locus) were cultured in serum-free primary culture with or without additional L-tyrosine (Tyr). The content of melanin was measured by high-performance liquid chromatography in the cultured melanocytes as well as culture supernatants in serum-free primary culture. L-Tyr was found to increase the content of pheomelanin in addition to eumelanin in cultured slaty melanocytes and cuture supernatants at all ages tested. The eumelanin and pheomelanin contents in culture supernatants were greater than in cultured melanocytes. The eumelanin and pheomelanin contents in culture supernatants from 7.5-day-old slaty melanocytes in the presence of L-Tyr were greater than those from wild-type melanocytes. These results suggest that the inhibition of eumelanin synthesis by the slaty mutation can be partly restored by the addition of excess L-Tyr. Eumelanin and pheomelanin may accumulate with difficulty in slaty melanocytes and be easily released from them during skin development. L-Tyr may stimulate this release.     

Melanosomal pH controls rate of melanogenesis, eumelanin/phaeomelanin ratio and melanosome maturation in melanocytes and melanoma cells.

The skin pigment melanin is produced in melanocytes in highly specialized organelles known as melanosomes. Melanosomes are related to the organelles of the endosomal/lysosomal pathway and can have a low internal pH. In the present study we have shown that melanin synthesis in human pigment cell lysates is maximal at pH 6.8. We therefore investigated the role of intramelanosomal pH as a possible control mechanism for melanogenesis. To do this we examined the effect of neutralizing melanosomal pH on tyrosinase activity and melanogenesis in 11 human melanocyte cultures and in 3 melanoma lines. All melanocyte cultures (9 of 9) from Caucasian skin as well as two melanoma cell lines with comparable melanogenic activity showed rapid (within 24 h) increases in melanogenesis in response to neutralization of melanosomal pH. Chemical analysis of total melanin indicated a preferential increase in eumelanin production. Electron microscopy revealed an accumulation of melanin and increased maturation of melanosomes in response to pH neutralization. In summary, our findings show that: (i) near neutral melanosomal pH is optimal for human tyrosinase activity and melanogenesis; (ii) melanin production in Caucasian melanocytes is suppressed by low melanosomal pH; (iii) the ratio of eumelanin/phaeomelanin production and maturation rate of melanosomes can be regulated by melanosomal pH. We conclude that melanosomal pH is an essential factor which regulates multiple stages of melanin production. Furthermore, since we have recently identified that pink locus product (P protein) mediates neutralization of melanosomal pH, we propose that P protein is a key control point for skin pigmentation. We would further propose that the wide variations in both constitutive and facultative skin pigmentation seen in the human population could be associated with the high degree of P-locus polymorphism.     

Keratinocytes control the proliferation and differentiation of cultured epidermal melanocytes from ultraviolet radiation B-induced pigmented spots in the dorsal skin of hairless mice

Long-term exposure of ultraviolet radiation B (UVB)-induced pigmented spots in the dorsal skin of hairless mice of Hos:(HR-1 X HR//De) F1. Previous study showed that the proliferative and differentiative activities of cultured epidermal melanoblasts/melanocytes from UVB-induced pigmented spots increased with the development of the pigmented spots. To determine whether the increase in the proliferative and differentiative activities of epidermal melanoblasts/melanocytes was brought about by direct changes in melanocytes, or by indirect changes in surrounding keratinocytes, pure cultured melanoblasts/melanocytes and keratinocytes were prepared and co-cultured in combination with control and irradiated mice in a serum-free culture medium. Keratinocytes from irradiated mice stimulated the proliferation and differentiation of both neonatal and adult non-irradiated melanoblasts/melanocytes more greatly than those from non-irradiated mice. In contrast, both non-irradiated and irradiated adult melanocytes proliferated and differentiated similarly when they were co-cultured with irradiated adult keratinocytes. These results suggest that the increased proliferative and differentiative activities of mouse epidermal melanocytes from UVB-induced pigmented spots are regulated by keratinocytes, rather than melanocytes.     

Quantitative approach of melanin in human cultured melanocytes

The purpose of the study was to establish an in vitro model for the quantification of the melanin produced in human melanocytes in culture. Melanin content in these cultured human melanocytes is determined by spectrofluorimetric assay. Fluorescence intensity is quantitatively related to the melanin content present in cultured melanocytes.     

Elutriation of human keratinocytes and melanocytes fromin vitro cultured epithelium

Summary Human epidermal keratinocytes grown in culture and at different stages of differentiation are shown to be viably separated by elutriation. A specific fraction enriched in melanocytes was obtained. Elutriation of cells obtained fromin vitro cultured epithlium could prove useful in studies concerning the biochemistry and molecular markers of cells isolated from normal epithelium and from different pathologies.     

The slaty mutation affects eumelanin and pheomelanin synthesis in mouse melanocytes

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.     

他卡西醇对人黑素细胞生物学性状的影响

目的:观察他卡西醇对体外培养的正常人黑素细胞增殖、黑素合成及酪氨酸酶(TYR)mRNA表达、酪氨酸酶相关蛋白-1(TRP-1)mRNA表达的影响,探讨他卡西醇治疗白癜风的机制.方法:体外黑素细胞培养,给予不同浓度他卡西醇(10-6、10-7、10-8、10-9、10-10mol/L)处理,用MTT法测定细胞增殖,碱裂解法测定黑素合成,多巴氧化法检测酪氨酸酶活性,半定量RT-PCR法检测TYR、TRP-1 mRNA的表达.结果:10-8 mol/L他卡西醇能刺激黑素细胞增殖,10-8、10-9 mol/L能促进黑素合成,10-9～10-6 mol/L的他卡西醇能显著增强正常人黑素细胞酪氨酸酶活性并上调TYR、TRP-1mRNA的表达.结论:他卡西醇可促进黑素细胞增殖,并提示该药物可能通过上调酪氨酸酶家族中关键基因TYR、TRP-1mRNA的表达来提高酪氨酸酶活性,最终增加黑素合成.     

Gangliosides of normal and neoplastic human melanocytes

The major ganglioside component isolated from diploid human melanocytes is sialosyllactosylceramide (GM3 86-91% of total sialic acid). The corresponding disialo derivative (GD3) is found as a minor component (2-6% of total sialic acid) in the membranes of these cells. In human melanoma cells, grown in tissue culture, GD3 is the predominant ganglioside component (48-63% of total sialic acid). Withdrawal of TPA from the culture medium of normal melanocytes or addition of TPA to the medium of melanoma cells had no significant effect on GM3/GD3 ratios. We conclude that the difference between the composition of gangliosides is related to the normal vs transformed phenotypes of melanocytes.     

Effect of cyclosporin A on melanogenesis in cultured human melanocytes

Cyclosporin A (CsA) is a widely used immunosuppressant. Reports on the effect of CsA on hyperpigmentation in patients appear inconsistent, and the effect of CsA on skin pigment cells (melanocytes) in vitro is unknown. We examined the effect of CsA on human melanocyte proliferation and melanogenesis in vitro. Melanocyte proliferation was dose-dependently inhibited by 0.1-10 microM CsA, with no effect on cell viability. Melanocytes incubated with 10 microM CsA for 6 days showed decreased pigmentation and tyrosinase activity. Western blot analysis using an anti-tyrosinase antibody revealed that CsA (0.1-10 microM) decreased tyrosinase protein levels in a dose-dependent manner. Northern blot analysis showed similar effects on tyrosinase mRNA levels. These effects of CsA on melanogenesis in vitro are not consistent with suggestions that systemic CsA therapy causes patient skin hyperpigmentation.     

Regulation of eumelanin/pheomelanin synthesis and visible pigmentation in melanocytes by ligands of the melanocortin 1 receptor

The production of melanin in the hair and skin is tightly regulated by the melanocortin 1 receptor (MC1R) whose activation is controlled by two secreted ligands, alpha-melanocyte stimulating hormone (alphaMSH) and agouti signal protein (ASP). As melanin is extremely stable, lasting years in biological tissues, the mechanism underlying the relatively rapid decrease in visible pigmentation elicited by ASP is of obvious interest. In this study, the effects of ASP and alphaMSH on the regulation of melanin synthesis and on visible pigmentation were assessed in normal murine melanocytes and were compared with the quick depigmenting effect of the tyrosinase inhibitor, phenylthiourea (PTU). alphaMSH increased pheomelanin levels prior to increasing eumelanin content over 4 days of treatment. Conversely, ASP switched off the pigment synthesis pathway, reducing eu- and pheo-melanin synthesis within 1 day of treatment that was proportional to the decrease in tyrosinase protein level and activity. These results demonstrate that the visible depigmentation of melanocytes induced by ASP does not require the degradation of existing melanin but rather is due to the dilution of existing melanin by melanocyte turnover, which emphasizes the importance of pigment distribution to visible color.     

Subcellular fractionation of cultured normal human melanocytes: New insights into the relationship of melanosomes with lysosomes and peroxisomes

In order to obtain information on the disputed nature of melanosomes a comparison was made between the localization of melanosomal markers with those of other well-defined subcellular organelles such as lysosomes and peroxisomes. The distribution of marker enzymes was studied using two different density gradient systems. i.e., Percoll and Nycodenz. Furthermore, the subcellular localization of various types of antigens was analyzed using indirect immunofluorescence and immuno-electron microscopy. All methods revealed the existence of partial co-localization of melanosomal and lysosomal proteins and different localization of peroxisomal markers. The results suggest that melanosomes may share a common origin with lysosomal structures.     

Tissue plasminogen activator is released into cultured medium by cultured human uveal melanocytes

Melanoma cells produce tissue plasminogen activator (t-PA) that plays an important role in tumor invasion and metastasis. The production of t-PA by normal human uveal melanocytes has not been reported previously. In order to explore this possibility, we studied the production of t-PA by cultured human uveal melanocytes and compared that with the production by cultured human uveal melanoma cells and epidermal melanocytes. Human adult uveal melanocytes were isolated and cultured from donor eyes. The cells were cultured in serum-free medium for 48 h and the conditioned medium then collected for the plasminogen activator (PA) activity assay. Free PA activity was tested in an amidolytic assay using a t-PA standard curve. PA type was identified by fibrinography and antihuman t-PA and urokinase plasminogen activator (u-PA) blocking antibodies. Free PA activity was found in the conditioned medium of normal melanocytes and melanoma cells. The predominant PA activity was t-PA. Normal uveal melanocytes produced more t-PA (3.23 +/- 0.73 IU/105 cells/24 h) than that of epidermal melanocytes (1.25 IU/105 cells/24 h) but much less than uveal melanoma cells (11.0 +/- 3.39 IU/105 cells/24 h). Western blot analysis revealed that most t-PA in conditioned media were one-chain t-PA with molecular weight of 69 kDa. Our study indicates that uveal melanocytes may contribute to the free t-PA activity previously found in aqueous humor and choroidal eye cup superfusions. Therefore, this function of uveal melanocytes may play a role in intraocular matrix remodeling, fibrinolysis and aqueous humor outflow.     

Coculture of human keratinocytes and melanocytes: differentiated melanocytes are physiologically organized in the basal layer of the cultured epithelium

Human epidermal keratinocytes differentiate in vitro into a stratified epithelium suitable for grafting on burned patients. In this paper, we show that differentiated melanocytes are present in the cultured epithelium. In particular, we have found that i) melanocytes proliferate in the same culture conditions that allow keratinocyte growth, ii) during the culture the ratio between keratinocytes and melanocytes tends to remain constant, iii) melanocytes organize into the basal layer of the cultured epithelium independently of the presence of dermis, develop dendritic arborizations with melanosome-containing processes and transfer melanosomes into keratinocyte cytoplasm.     

Ultraviolet radiation directly induces pigment production by cultured human melanocytes

In humans the major stimulus for cutaneous pigmentation is ultraviolet radiation (UVR). Little is known about the mechanism underlying this response, in part because of the complexity of interactions in whole epidermis. Using a recently developed culture system, human melanocytes were exposed daily to a physiologic range of UVR doses from a solar simulator. Responses were determined 24 hours after the last exposure. There was a dose-related increase in melanin content per cell and uptake of 14C-DOPA, accompanied by growth inhibition. Cells from donors of different racial origin gave proportionately similar increases in melanin, although there were approximately tenfold differences in basal values. Light and electron microscopy revealed UVR-stimulated increases in dendricity as well as melanosome number and degree of melanization, analogous to the well-recognized melanocyte changes following sun exposure of intact skin. Similar responses were seen with Cloudman S91 melanoma cells, although this murine cell line required lower UVR dosages and fewer exposures for maximal stimulation. These data establish that UVR is capable of directly stimulating melanogenesis. Because cyclic AMP elevation has been associated in some settings with increased pigment production by cultured melanocytes, preliminary experiments were conducted to see if the effects of UVR were mediated by cAMP. Both alpha-MSH and isobutylmethylxanthine (IBMX), as positive controls, caused a fourfold increase in cAMP level in human melanocytes and/or S91 cells, but following a dose of UVR sufficient to stimulate pigment production there was no change in cAMP level up to 4 hours after exposure. Thus it appears that the UVR-induced melanogenesis is mediated by cAMP-independent mechanisms.     

Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio

The skin constitutive pigmentation is given by the amount of melanin pigment, its relative composition (eu/pheomelanin) and distribution within the epidermis, and is largely responsible for the sensitivity to UV exposure. Nevertheless, a precise knowledge of melanins in human skin is lacking. We characterized the melanin content of human breast skin samples with variable pigmentations rigorously classified through the Individual Typology Angle (ITA) by image analysis, spectrophotometry after solubilization with Soluene‐350 and high‐performance liquid chromatography (HPLC) after chemical degradation. ITA and total melanin content were found correlated, ITA and PTCA (degradation product of DHICA melanin), and TTCA (degradation product of benzothiazole‐type pheomelanin) as well but not 4‐AHP (degradation product of benzothiazine‐type pheomelanin). Results revealed that human epidermis comprises approximately 74% of eumelanin and 26% pheomelanin, regardless of the degree of pigmentation. They also confirm the low content of photoprotective eumelanin among lighter skins thereby explaining the higher sensitivity toward UV exposure.     

Tumor necrosis factor enhances GD3 ganglioside expression in cultured human melanocytes

Human skin melanocytes and melanocytes cultured in vitro express GM3 ganglioside almost exclusively, whereas malignant melanomas express high levels of both GM3 and GD3. We now show that treatment of cultured melanocytes with tumor necrosis factor-alpha, particularly in the presence of tetradecanoylphorbol-13-acetate, results in a change in morphology from spindle-shaped to epithelioid and greatly enhanced expression of GD3 ganglioside. This effect is specific and no other ganglioside is affected, except that GM3 expression (which is already high) is also increased. In contrast, these agents did not enhance the already high expression of GD3 on melanoma cells. This result provides an example of the plasticity of glycolipid expression in mammalian cells and their susceptibility to the influence of biological agents.