Role of Dermal Factors Involved in Regulating the Melanin and Melanogenesis of Mammalian Melanocytes in Normal and Abnormal Skin.

Mammalian melanin is produced in melanocytes and accumulated in melanosomes. Melanogenesis is supported by many factors derived from the surrounding tissue environment, such as the epidermis, dermis, and subcutaneous tissue, in addition to numerous melanogenesis-related genes. The roles of these genes have been fully investigated and the molecular analysis has been performed. Moreover, the role of paracrine factors derived from epidermis has also been studied. However, the role of dermis has not been fully studied. Thus, in this review, dermis-derived factors including soluble and insoluble components were overviewed and discussed in normal and abnormal circumstances. Dermal factors play an important role in the regulation of melanogenesis in the normal and abnormal mammalian skin.

Reduced Elastin Fibers and Melanocyte Loss in Vitiliginous Skin Are Restored after Repigmentation by Phototherapy and/or Autologous Minigraft Transplantation.

Vitiligo is a hypopigmentation disease characterized by melanocyte death in the human epidermis. However, the mechanism of vitiligo development and repigmentation is largely unknown. Dermal fiber components might play an important role in vitiligo development and repigmentation. Indeed, our preliminary study demonstrated that elastin fibers were decreased in vitiliginous skin, suggesting that the elastin fiber is one of the factors involved in vitiligo development and repigmentation. To confirm our hypothesis, we investigated whether elastin fibers can be restored after treatment using phototherapy and/or autologous skin transplantation. Punch biopsies from 14 patients of stable nonsegmental vitiligo vulgaris were collected from nonlesional, lesional, and repigmented skin, and processed to dopa and combined dopa-premelanin reactions. Melanocytes positive to the dopa reaction and melanoblasts/melanocytes positive to the combined dopa-premelanin reaction were surveyed. Moreover, elastin fibers were detected by Victoria blue staining. Numerous melanocytes and melanoblasts were observed in the epidermis of repigmented skin after the treatment. Moreover, in the dermis of repigmented skin, elastin fibers were completely recovered or even upregulated. These results suggest that melanocyte loss in the vitiliginous skin, as well as melanocyte differentiation in repigmented skin, may be at least in part regulated by elastin fibers in the dermis.

The human melanocyte and melanoblast populations per unit area of epidermis in the rete ridge are greater than in the inter-rete ridge.

OBJECTIVE: In human skin, melanocytes and melanoblasts are mostly located in the epidermis in addition to hair follicles. In hairy skin such as mouse skin, the inter-follicular epidermis is generally flat. In contrast, in human skin, the epidermis is wavy and possesses well-developed rete ridges. It is not well understood what difference exists in cell function between melanocytes present in the inter-rete ridge and those in the rete ridge. To clarify this problem, we first tried to determine the melanocyte and melanoblast populations per unit area of epidermis both in the rete ridge and inter-rete ridge epidermis. METHODS: Thirty-eight samples from normal skin sites of 28 patients (14 males and 14 females) aged from 5 to 76 years were fixed and processed to dopa and combined dopa-premelanin reactions. The numbers of cells positive to the dopa (melanocytes) and combined dopa-premelanin (melanoblasts and melanocytes) reactions were scored. RESULTS: The average melanocyte population/0.1 mm2 of the inter-rete ridge was 74 cells, whereas that of the rete ridge was 147 cells. Moreover, the average melanoblast population/0.1 mm2 in the inter-rete ridge was 43 cells, whereas that of the rete ridge was 131 cells. The melanoblast population in the rete ridge differed between female and male, but the melanocyte populations in the rete ridge and inter-rete ridge and the melanoblast population in the inter-rete ridge did not differ between female and male. However, no age difference in the melanocyte and melanoblast populations was observed between the rete ridge and inter-rete ridge. CONCLUSION: Human epidermal melanocytes and melanoblasts in the rete ridge exist more numerously than in the inter-rete ridge.

OBJECTIF: dans la peau humaine, les mélanocytes et les mélanoblastes sont principalement situés dans l'épiderme ainsi que dans les follicules pileux. Dans la peau poilue, comme la peau de souris, l'épiderme interfolliculaire est généralement plat. En revanche, dans la peau humaine, l'épiderme a un aspect ondulé et possède des crêtes épidermiques bien développées. On ne comprend pas bien la différence entre la fonction cellulaire des mélanocytes présents dans la crête inter-épidermique et celle des mélanocytes présents dans la crête épidermique. Afin de clarifier ce problème, nous avons d'abord essayé de déterminer les populations mélanocytaires et mélanoblastiques par unité de surface de l'épiderme, à la fois dans l'épiderme de la crête épidermique et dans celui de la crête inter-épidermique. MÉTHODES: trente-huit échantillons provenant de sites cutanés normaux de 28 patients (14 hommes et 14 femmes), âgés de 5 à 76 ans, ont été fixés et traités pour des réactions à la dopa et à l'association dopa-prémélanine. Le nombre de cellules positives aux réactions à la dopa (mélanocytes) et à l'association dopa-prémélanine (mélanoblastes et mélanocytes) a été évalué. RÉSULTATS: la population mélanocytaire moyenne/0,1 mm2 de la crête inter-épidermique était de 74 cellules, alors que celle de la crête épidermique était de 147 cellules. De plus, la population mélanoblastique moyenne/0,1 mm2 de la crête inter-épidermique était de 43 cellules, alors que celle de la crête épidermique était de 131 cellules. La population mélanoblastique dans la crête épidermique était différente entre les femmes et les hommes, mais les populations mélanocytaires dans la crête épidermique et la crête inter-épidermique, ainsi que la population mélanoblastique dans la crête inter-épidermique, n'étaient pas différentes entre les femmes et les hommes. Cependant, aucune différence liée à l'âge n'a été observée entre la crête épidermique et la crête inter-épidermique dans les populations mélanocytaires et mélanoblastiques. CONCLUSION: chez l'homme, les mélanocytes et les mélanoblastes épidermiques sont plus nombreux dans la crête épidermique que dans la crête inter-épidermique.

Improved HPLC Conditions to Determine Eumelanin and Pheomelanin Contents in Biological Samples Using an Ion Pair Reagent.

Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin and pheomelanin, two major classes of melanin pigments, affords pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) from eumelanin and thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA) from pheomelanin. Quantification of these five markers by HPLC provides useful information on the quantity and structural diversity of melanins in various biological samples. HPLC analysis of these markers using the original method of 0.1 M potassium phosphate buffer (pH 2.1):methanol = 99:1 (85:15 for PTeCA) on a reversed-phase column had some problems, including the short lifetime of the column and, except for the major eumelanin marker PTCA, other markers were occasionally overlapped by interfering peaks in samples containing only trace levels of these markers. These problems can be overcome by the addition of an ion pair reagent for anions, such as tetra-n-butylammonium bromide (1 mM), to retard the elution of di-, tri- and tetra-carboxylic acids. The methanol concentration was increased to 17% (30% for PTeCA) and the linearity, reproducibility, and recovery of the markers with this improved method is good to excellent. This improved HPLC method was compared to the original method using synthetic melanins, mouse hair, human hair, and human epidermal samples. In addition to PTCA, TTCA, a major marker for pheomelanin, showed excellent correlations between both HPLC methods. The other markers showed an attenuation of the interfering peaks with the improved method. We recommend this improved HPLC method for the quantitative analysis of melanin markers following AHPO because of its simplicity, accuracy, and reproducibility.

Histochemical study of the distribution of epidermal melanoblasts and melanocytes in Asian human skin.

INTRODUCTION: The classical study revealed the melanocyte density in the epidermis of many skin sites of humans. However, these data were obtained by counting melanocytes using dihydroxyphenylalanine (dopa)-treated epidermal sheets. Since rete ridges are developed well in human epidermis, there is a concern about the accuracy of these data. The accurate counting of the melanocyte density in the epidermis including the rete ridges should be performed using histological sections after the dopa treatment. Moreover, it is not known well how many melanoblasts are present in Asian epidermis. The aim of this study was to count the accurate number of melanocytes and melanoblasts. METHODS: Normal skin sites of 9- to 77-year-old patients were fixed with buffered formalin and processed to the dopa and combined dopa-premelanin reactions. The numbers of cells positive to the dopa reaction (melanocytes) and to the combined dopa-premelanin reaction (melanoblasts and melanocytes) were scored. RESULTS: In the skin of arms, legs, back, and belly, similar density (approximately 110-120 cells/0.1 mm2 ) of melanocytes was observed, whereas in the skin of scalp, melanocyte density was much lower (approximately 70 cells/0.1 mm2 ). By contrast, the melanoblast density did not differ between skin sites (approximately 100 cells/0.1 mm2 ). CONCLUSIONS: These results suggest that the melanocyte density does not differ between skin sites except the scalp skin, and a certain number of melanoblasts are present in each skin site of Asian. Melanoblasts seem to be required for producing new melanocytes required to maintain epidermal homeostasis.

The Pro-Oxidant Activity of Pheomelanin is Significantly Enhanced by UVA Irradiation: Benzothiazole Moieties Are More Reactive than Benzothiazine Moieties.

It is generally considered that eumelanin (EM) is photoprotective while pheomelanin (PM) is phototoxic. A recent study using a mouse model demonstrated that PM produces reactive oxygen species (ROS) that cause DNA damage and eventually lead to melanomagenesis. A biochemical study showed that PM possesses a pro-oxidant activity. PM consists of benzothiazine (BT) and benzothiazole (BZ) moieties, BT moieties being transformed to BZ moieties by heat or light. In this study, we compared the effects of ultraviolet A (UVA) irradiation using synthetic PMs with different BT to BZ ratios and using various coat color mouse hairs. We found that UVA irradiation of BZ-PM increased glutathione (GSH) depletion and generated more H2O2 than UVA irradiation of BT-PM. Non-irradiated controls did not exhibit strong pro-oxidant activities. Upon UVA irradiation, yellow mouse hairs oxidized GSH and produced H2O2 faster than black or albino mouse hairs. Next, to examine the mechanism of the pro-oxidant activity of BT-PM and BZ-PM, we examined the pro-oxidant activities of 7-(2-amino-2-carboxyethyl)-dihydro-1,4-benzothiazine-3-carboxylic acid (DHBTCA) and 6-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole (BZ-AA) as BT and BZ monomers, respectively. Their pro-oxidant activities were similar, but a large difference was seen in the effects of ROS scavengers, which suggests that the redox reactions may proceed via singlet oxygen in BZ-AA and via superoxide anions in DHBTCA. These results show that UVA enhances the pro-oxidant activity of PM, in particular BZ-PM.

Multipotency of melanoblasts isolated from murine skin depends on the Notch signal.

BACKGROUND: Melanoblasts (MBs), derived from neural crest cells, only differentiate into melanocytes (Ms) in vivo. We previously showed that MBs isolated from mouse skin were multipotent, generating neurons (Ns) and glial cells (Gs) together with Ms. Using Sox10-IRES-Venus mice and mouse embryonic stem cells, we investigated how MBs expressed their multipotency. RESULTS: MBs generated colonies containing Ns, Gs, and Ms in the presence of ST2 stromal cells, but they generated only M colonies when incubated with keratinocytes or ST2 culture supernatant, thus showing that MBs required contact with ST2 stromal cells for expression of their multipotency. Notch signaling was shown to be one of the important cues for the maintenance and differentiation of MBs through cell-cell contact. When Notch signaling was inhibited, MBs mainly generated colonies that contained just one type of cells, Ns, Gs, or Ms; the number of colonies containing two or three types of cells markedly decreased even on ST2 stromal cells, showing restriction of their differentiation potency. Whereas when Notch signaling was activated, the number of colonies containing two or three types of cells increased, indicating that their multipotency had been maintained. CONCLUSIONS: Our results demonstrate that Notch signaling played novel roles in MB multipotency.

Regeneration of Murine Hair Follicles is Inhibited by Low-Dose-Rate Gamma Irradiation.

To determine whether the effects of low-dose-rate gamma (γ) irradiation are identifiable in the regeneration of murine hair follicles, we irradiated whole bodies of C57BL/10JHir mice in the first telogen phase of the hair cycle with 137Cs γ-rays. The mice were examined for effects on hair follicles, including number, morphology, and pigmentation in the second anagen phase. Effects of γ-radiation on melanocyte stem cells were also investigated by the indirect immunolabeling of tyrosinase-related protein 2 (TRP2). Irradiated skin showed a decrease in hair follicle density and the induction of curved hair follicles along with the presence of white hairs and hypopigmented hair bulbs. There was a small, but not significant, change in the number of TRP2-positive melanocyte stem cells in the hair bulge region of the irradiated skin. These results suggest that low-dose rate γ-irradiation does not deplete melanocyte stem cells, but can damage stem cells and progenitors for both keratinocytes and melanocytes, thereby affecting the structure and pigmentation of regenerated hair follicles in the 2nd anagen phase.

The mouse brown (b/Tyrp1(b) ) allele does not affect pheomelanin synthesis in mice.

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. Melanosomes of cultured brown melanocytes were mostly spherical stage III melanosomes with granular depositions of pigments, whereas those of cultured black melanocytes were mostly stage IV ellipsoidal melanosomes with pigment depositions in intraluminal fibrils. Chemical analysis of melanin present in dorsal hairs of 5-week-old mice from the F2 generation between brown and recessive yellow (B10-Mc1r(e)/Mc1r(e) ) or agouti (B10-A/A) mice showed that eumelanin content was greatly decreased in brown and brown agouti (cinnamon) mice, whereas pheomelanin contents in brown recessive yellow and cinnamon mice did not differ from the corresponding Tyrp1(+)/- mice. These results suggest that the brown allele greatly inhibits eumelanin, but not pheomelanin synthesis.

Reduced proliferative and differentiative activity of mouse pink-eyed dilution melanoblasts is related to apoptosis.

The mouse pink-eyed dilution (p) locus is known to control the melanin content, melanosome morphology, and tyrosinase activity in melanocytes. However, it is not well known whether the p allele is involved in regulating melanocyte proliferation, differentiation, and death. The aim of this study is to investigate in detail the role of the p allele in melanocyte proliferation, differentiation, and death using a cell culture system. The epidermal cell suspensions of the neonatal dorsal skin derived from wild type mice at the p locus (black, C57BL/10JHir-P/P) and their congenic mutant (pink-eyed dilution, C57BL/10JHir-p/p) were cultured with serum-free melanoblast-proliferation medium (MDMDF) and melanocyte-proliferation medium (MDMD). The proliferation and differentiation of p/p melanoblasts in MDMDF or MDMD were greatly inhibited compared with those of P/P melanoblasts and melanocytes. It is possible that apoptosis is related to the reduced proliferative and differentiative activity of p/p melanoblasts/melanocytes. The addition of apoptosis-inhibitors, such as caspase-9 inhibitor (C9I) and Bax-inhibiting peptide (BIP) into MDMDF or MDMD stimulated the proliferation and differentiation of p/p melanoblasts. In contrast, in P/P melanoblasts and melanocytes, C9I and BIP failed to stimulate their proliferation and differentiation. The number of apoptotic keratinocytes and melanoblasts/melanocytes in p/p mice was greater than in P/P mice. Moreover, expression of C9 and Bax in keratinocytes and melanoblasts/melanocytes in p/p mice was greater than in P/P mice. These results suggest that the increased apoptosis in keratinocytes and melanoblasts/melanocytes is related to the reduced proliferative and differentiative activity of p/p melanoblasts.

A new mutation of mouse ruby-eye 2, ru2(d)/Hps5(ru2-d) inhibits eumelanin synthesis but stimulates pheomelanin synthesis in melanocytes.

We previously demonstrated that a novel mutation, characterized by light-colored coats and ruby eyes, which occurred spontaneously in mice in our laboratory, exhibited deletion in the Hps5 gene (ru2(d)/Hps5(ru2-d)). To clarify the mechanism of this hypopigmentation, the characteristics of the neonatal development of ru2(d)/ru2(d) melanocytes were investigated in detail with special reference to those of +/+ melanocytes. In ru2(d)/ru2(d) mice, there were fewer epidermal melanocytes than in +/+ mice, whereas there was no difference in numbers of epidermal melanoblasts in +/+ and ru2(d)/ru2(d)mice, both in dorsal and ventral skin. Epidermal melanocytes with increased dopa-melanin deposition and dendritogenesis were greatly increased by injecting L-Tyr subcutaneously into newborn ru2(d)/ru2(d) mice. The eumelanin content in the epidermis and dermis in postnatal ru2(d)/ru2(d) mice was much lower than in +/+ mice, whereas similar pheomelanin content was observed 5.5 or 7.5 days after birth both in dorsal and ventral skins. Moreover, the eumelanin content in the dorsal and ventral hairs in 5-week-old ru2(d)/ru2(d) mice was much lower than in +/+ mice, whereas pheomelanin content was two to four times greater than in +/+ mice. These results suggest that the ru2(d) allele suppresses the differentiation of melanocytes through the inhibition of eumelanin synthesis, but stimulates pheomelanin synthesis in melanocytes.

Neural crest cells retain their capability for multipotential differentiation even after lineage-restricted stages.

Multipotency of neural crest cells (NC cells) is thought to be a transient phase at the early stage of their generation; after NC cells emerge from the neural tube, they are specified into the lineage-restricted precursors. We analyzed the differentiation of early-stage NC-like cells derived from Sox10-IRES-Venus ES cells, where the expression of Sox10 can be visualized with a fluorescent protein. Unexpectedly, both the Sox10+/Kit- cells and the Sox10+/Kit+ cells, which were restricted in vivo to the neuron (N)-glial cell (G) lineage and melanocyte (M) lineage, respectively, generated N, G, and M, showing that they retain multipotency. We generated mice from the Sox10-IRES-Venus ES cells and analyzed the differentiation of their NC cells. Both the Sox10+/Kit- cells and Sox10+/Kit+ cells isolated from these mice formed colonies containing N, G, and M, showing that they are also multipotent. These findings suggest that NC cells retain multipotency even after the initial lineage-restricted stages.

Effects of low-dose γ-rays on the embryonic development of mouse melanoblasts and melanocytes in the epidermis and hair bulbs.

The effects of low-dose γ-rays on the embryonic development of animal cells are not well studied. The mouse melanocyte is a good model to study the effects of low-dose γ-rays on the development of animal cells, as it possesses visible pigment (melanin) as a differentiation marker. The aim of this study is to investigate in detail the effects of low-dose γ-rays on embryonic development of mouse melanoblasts and melanocytes in the epidermis and hair bulbs at cellular level. Pregnant females of C57BL/10J mice at nine days of gestation were whole-body irradiated with a single acute dose of γrays (0.1, 0.25, 0.5, and 0.75 Gy), and the effects of γ-rays were studied by scoring changes in the development of epidermal melanoblasts and melanocytes, hair follicles, and hair bulb melanocytes at 18 days in gestation. The number of epidermal melanoblasts and melanocytes, hair follicles, and hair bulb melanocytes in the dorsal and ventral skins was markedly decreased even at 0.1 Gy-treated embryos (P < 0.001), and gradually decreased as dose increased. The effects on the ventral skin were greater than those on the dorsal skin. The dramatic reduction in the number of melanocytes compared to melanoblasts was observed in the ventral skin, but not in the dorsal skin. These results suggest that low-dose γ-rays provoke the death of melanoblasts and melanocytes, or inhibit the proliferation and differentiation of melanoblasts and melanocytes, even at the low dose.

A novel deletion mutation of mouse ruby-eye 2 named ru2(d)/Hps5(ru2-d) inhibits melanocyte differentiation and its impaired differentiation is rescued by L-tyrosine.

In our laboratory, a single autosomal recessive mutation in a phenotype similar to ruby-eye (ru/Hps6(ru)) or ruby-eye 2 (ru2/Hps5(ru2)) spontaneously occurred in siblings of C57BL/10JHir (+/+, black) mice in 2006. RT-PCR analysis revealed that this novel mutation, named ru2(d)/Hps5(ru2-d), exhibited frameshift by 997G deletion in the Hps5 gene. To clarify the mechanism of the hypopigmentation, the characteristics of proliferation and differentiation of ru2(d)/ru2(d) epidermal melanoblasts and melanocytes cultured in a serum-free medium were investigated. The proliferation of ru2(d)/ru2(d) melanoblasts and melanocytes did not differ from that of +/+ melanoblasts and melanocytes. However, the differentiation of ru2(d)/ru2(d) melanocytes was greatly inhibited. Tyrosinase (TYR) activity, expression of TYR, TYR-related protein 1 (TRP1) and TRP2 (dopachrome tautomerase, DCT), eumelanin synthesis, and the number of stage IV melanosomes markedly decreased in ru2(d)/ru2(d) melanocytes. However, excess L-tyrosine (Tyr) added to culture media from initiation of the primary culture rescued the reduced differentiation through increase in TYR activity, expression of TYR, TRP1, TRP2 and Kit, eumelanin synthesis, and stage IV melanosomes. L-Tyr injected into ru2(d)/ru2(d) mice also stimulated melanocyte differentiation. These results suggest that the ru2(d) allele inhibits melanocyte differentiation, and that its impaired differentiation is rescued by excess Tyr.

Stimulation of the proliferation and differentiation of skin cells by ferrous ferric chloride from a distance.

Ferrous ferric chloride (FFC) is a distinct form of aqueous iron composed of a complex of ferrous chloride and ferric chloride that participates in both oxidation and reduction reactions, and stimulates the proliferation and differentiation of mammalian keratinocytes, melanocytes, and fibroblasts. However, it is not known whether FFC can stimulate their proliferation and differentiation without being added into culture media or painted on the skin. This study aims to clarify whether FFC can stimulate their proliferation and differentiation from a distance without being added to culture media. In this study, FFC-containing skin lotions were painted under the culture dishes (1 mm away from cells) or on the top of the covers of 1 to 5 polystyrene culture dishes (1 to 5 cm away) and tested for their proliferation- and differentiation-stimulating effects. FFC lotions stimulated the proliferation and differentiation of human keratinocytes, melanocytes, and fibroblasts from a distance of 1 mm to 1 cm. However, FFC lotions failed to stimulate the proliferation and differentiation of melanocytes from distances of 2 to 5 cm. Results using Teflon covers were similar to those of polystyrene covers. Moreover, the effects of FFC lotions painted on the top of the Teflon covers were completely lost by lead disks. These results suggest that FFC can stimulate the proliferation and differentiation of skin cells from a distance of 1 cm without being added into culture media through physical factors rather than chemical factors.

Estrogen increases hair pigmentation in female recessive yellow mice.

Murine recessive yellow (Mc1r(e)) is a loss-of-function mutation in the receptor for alpha-melanocyte-stimulating hormone, melanocortin receptor 1 (Mc1r), and results in a yellow coat by inducing pheomelanin synthesis in hair follicular melanocytes. We previously showed that eumelanin and pheomelanin content in dorsal hair in female Mc1r(e)/Mc1r(e) mice 5 weeks after birth was greater than that in male mice. To better understand the sex difference in hair pigmentation, estrogen, progesterone, and androgen were injected subcutaneously to newborn mice, and the effects of these sex hormones on hair pigmentation at 8.5 days were investigated. Although these sex hormones failed to affect the ratio of pheomelanic to eumelanic hair, they increased total hair pigmentation. Chemical analyses showed that total melanin content was increased by a low dose of estrogen in female mice. Moreover, estrogen increased pheomelanin content in female hair, whereas the hormone greatly decreased both eumelanin and pheomelanin content in male hair. High doses of progesterone, in contrast, increased total melanin content in both female and male hair. Moreover, a high dose of androgen increased total melanin content exclusively in male hair. These results suggest that estrogen is the main factor in determining the higher content of eumelanin and pheomelanin in female hair of Mc1r(e)/Mc1r(e) mice.

Ferrous ferric chloride stimulates the skin cell function and hair growth in mice.

Ferrous ferric chloride (FFC) is a distinct form of aqueous iron composed of a complex of ferrous chloride and ferric chloride that participates in both oxidation and reduction reactions. The author's previous study showed that the supplementation of culture medium with FFC stimulated the proliferation and differentiation of keratinocytes and melanocytes in newborn mice. FFC also stimulated the proliferation of cultured human keratinocytes, fibroblasts, and melanocytes. However, it is not known whether FFC stimulates the proliferation and differentiation of mammalian skin cells as well as hair growth in vivo. To answer this question, FFC-containing skin lotions (FFC Super Essence Plain and Moisture Type, Akatsuka Co.) were painted on the dorsal skin of newborn C57BL/10JHir (B10) mice and tested for their proliferation- and differentiation-stimulating effects on keratinocytes, fibroblasts, and melanocytes as well as for their stimulating effects on the hair growth. This treatment stimulated the proliferation and differentiation of epidermal keratinocytes, dermal fibroblasts, and epidermal and dermal melanocytes in the skin as well as hair growth. From 2 to 3 weeks after birth B10 mice generally lose their hairs except those on the head at the telogen stage of the hair growth cycle due to the expression of the alopecia. The treatment with FFC lotions markedly inhibited the alopecia hair-loss. These results suggest that FFC can stimulate the proliferation and differentiation of keratinocytes, fibroblasts, and melanocytes in the skin as well as the hair growth, and, in addition, can inhibit the alopecia hair-loss.

Activation of melanoblasts and melanocytes after treatment with monochromatic excimer light and narrowband-ultraviolet B of skin of vitiligo patients.

BACKGROUND: Vitiligo is a hypopigmentation disease characterized by achromatic or hypochromatic macules in several sites of human skin. Although monochromatic excimer light (MEL) and narrowband-ultraviolet B (NB-UVB) have recently been used for efficacious treatment of vitiligo, the repigmentation process and role of melanocytes/melanoblasts are not well known. METHODS: White macules of 28 vitiligo patients were repeatedly exposed with MEL/NB-UVB. After the exposures, punch biopsies of the center and edge of the lesional skin in addition to normal skin were fixed and processed to dopa and combined dopa-premelanin reactions. Melanocytes positive to the dopa reaction and melanoblasts/melanocytes positive to the combined dopa-premelanin reaction were surveyed. RESULTS: Two repigmentation patterns were observed; one was "marginal" and the other was "perifollicular." Although the frequency of the former pattern was much lower than that of the latter pattern, no differences were observed in average percentage of repigmentation and the number of melanocytes and melanoblasts observed at the center and edge of the lesional epidermis. CONCLUSIONS: These results suggest that exposures with MEL/NB-UVB induce the marginal or perifollicular repigmentation pattern in the white macules, though the two patterns produce no differences in the distribution and density of melanoblasts/melanocytes in the lesional epidermis.

Mitochondria are more numerous and smaller in pink-eyed dilution melanoblasts and melanocytes than in wild-type melanocytes in the neonatal mouse epidermis.

Abstract The mouse pink-eyed dilution (p) locus is known to control the melanin content in melanocytes. However, it was not known whether the p gene is involved in regulating the proliferation and differentation of melanocytes during development, especially the biogenesis of melanosomes and other organelles. Epidermal cell suspensions of neonatal dorsal skin derived from mice wild type for the p locus (black, C57BL/10JHir-P/P) and their congenic mutant phenotype (pink-eyed dilution, C57BL/10JHir-p/p) were cultured in serum-free melanocyte-proliferation medium (MDMD). The supplement of additional L-tyrosine (Tyr) into the MDMD stimulated the differentiation of p/p melanoblasts into melanocytes. Electron microscopy revealed that in p/p melanoblasts and melanocytes treated with L-Tyr, the number of stage II and III melanosomes dramatically increased. Moreover, p/p melanoblasts possessed smaller but more numerous mitochondria than P/P melanocytes. The treatment of p/p melanoblasts and melanocytes with L-Tyr decreased the number of mitochondria. The supplement of 2, 4-dinitrophenol (DNP), an inhibitor of mitochondrial function, into the MDMD stimulated both the proliferation and differentiation of p/p melanoblasts. Simultaneous treatment of DNP and L-Tyr dramatically stimulated the differetiation of p/p melanocytes. These results suggest that L-Tyr and some unknown factors related to mitochondrial function may influence the differentiation of melanoblasts in the epidermis of p/p mice.