Effects of hydroxybenzyl alcohols on melanogenesis in melanocyte-keratinocyte co-culture and monolayer culture of melanocytes

In mammalian skin, melanocyte proliferation and melanogenesis can be stimulated by keratinocytes, fibroblasts and other regulatory factors. To determine whether hydroxybenzyl alcohols (HBAs) show more inhibitory in melanocytes cultured alone or in melanocytes co-cultured with keratinocytes, we developed a murine melanocyte-keratinocyte co-culture model to investigate the pigmentation regulators in company with other melanogenic inhibitors and stimulators. It was found that the effects of HBAs and melanogenic factors were more evident in melanocytes co-cultured with keratinocytes. Keratinocytes may play a synergistic role in melanocyte melanogenesis and influence the pigment production. The tests in the co-culture model also imply that the inhibitory effects of HBAs on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity. HBAs showed a low cytotoxicity. The eventual results proved that HBAs are promising and safe agents for skin whitening in melanocyte alone and in co-culture systems. The co-culture model provides a more physiologically realistic condition to study the interaction between melanocytes and keratinocytes, which enables a reliable screening system for depigmenting compounds.     

Wnt inhibitory factor (WIF)‐1 promotes melanogenesis in normal human melanocytes

Wnt signaling plays a role in the differentiation as well as the development of melanocytes. Using a microarray analysis, hyperpigmentary skin of melasma expressed high levels of Wnt inhibitory factor‐1 (WIF‐1) compared with perilesional normal skin. In this study, the expression and functional roles of WIF‐1 on melanocytes were investigated. WIF‐1 was expressed both in the melanocytes of normal human skin and in cultured melanocytes. The upregulation of WIF‐1 on cultured normal human melanocytes significantly induced expressions of MITF and tyrosinase, which were associated with increased melanin content and tyrosinase activity. Consistent with the stimulatory effect of WIF‐1, WIF‐1 siRNA reduced melanogenesis in the cells. Moreover, WIF‐1 increases pigmentation in melanocytes co‐cultured with WIF‐1‐overexpressed fibroblasts and of organ‐cultured human skin. These findings suggest that melanocytes express WIF‐1 constitutively in vivo and in vitro and that WIF‐1 promotes melanogenesis in normal human melanocytes.     

Pleiotrophin inhibits melanogenesis via Erk1/2‐MITF signaling in normal human melanocytes

Pleiotrophin (PTN) is a secreted heparin‐binding protein that is involved in various biological functions of cell growth and differentiation. Little is known about the effects of PTN on the melanocyte function and skin pigmentation. In this study, we investigated whether PTN would affect melanogenesis. PTN was expressed in melanocytes and fibroblasts of human skin. Transfection studies revealed that PTN decreased melanogenesis, probably through MITF degradation via Erk1/2 activation in melanocytes. The inhibitory action of PTN in pigmentation was further confirmed in ex vivo cultured skin and in the melanocytes cocultured with fibroblasts. These findings suggest that PTN is a crucial factor for the regulation of melanogenesis in the skin.     

Normal murine melanocytes in culture

Summary A major obstacle to applying the techniques of molecular biology to the genetics and cell biology of pigmentation has been our inability to grow normal murine melanocytes in culture. We report here the establishment and characterization of continuously proliferating cultures of cutaneous pigment cells from seven strains of mice. Melanocytes were grown from the dermis of newborn mice in medium containing 12-0-tetradecanoyl-13-phorbol-acetate; a substance, such as melanotropin, that raises intracellular levels of cyclic AMP; and an extract made from human placenta. This work was supported by Grant R01 CA04679 from the U.S. National Institutes of Health and a fellowship to Dr. A. Tamura from Mr. and Mrs. Allen Locklin. The chromosome studies were carried out in the laboratory of Dr. Uta Francke, Department of Human Genetics, Yale University. JCM was supported by NIH contract number N01-CP-21037.     

Kaempferol promotes melanogenesis and reduces oxidative stress in PIG1 normal human skin melanocytes

Vitiligo is an autoimmune disease characterized by depigmentation. Kaempferol is a flavonoid compound with broad anti-inflammatory and antioxidant properties. The purpose of this study was to investigate the effect of kaempferol on melanogenesis in PIG1 normal human skin melanocytes and its response to oxidative stress. The effect of kaempferol on melanin synthesis in PIG1 normal human skin melanocytes was explored by measuring tyrosinase activity, melanin content, mRNA and protein expression of key enzymes and expression of related pathway proteins. The effects of kaempferol pretreatment on cell viability, apoptosis, ROS level and HO-1 protein level under H₂O₂ stimulation were explored. When treated with kaempferol, the tyrosinase activity and melanin content of PIG1 cells increased, the mRNA and protein expressions of TYR, TRP1, TRP2 and MITF increased, and the phosphorylation level of ERK1/2 increased. Upon the stimulation of H₂O₂, kaempferol reduced the production of ROS, decreased apoptosis and increased the protein expression of HO-1 in PIG1 cells. In addition, kaempferol inhibited oxidative stress-induced melanin reduction and promoted melanin synthesis in PIG1 cells and protected against H₂O₂-induced oxidative stress damage.     

Effect of keratinocytes on regulation of melanogenesis in culture of melanocytes

Melanocytes are the melanin-producing cells by melanogenesis, and the pigment melanin is primarily responsible for the color of skin. These cells contain dendrites that are in close contact with neighboring keratinocytes. Keratinocytes produce and secrete factors that regulate the proliferation and melanogenesis of melanocytes in vitro. Therefore, adopting only melanocyte pure culture may not clearly reflect the skin physiology in vivo. In this study, we applied a two-culture model using melanocytes and keratinocytes from human skin, such as melanocyte pure culture and melanocyte co-culture with keratinocyte. And then, there was compared the responses of melanocytes under different culture conditions (treatment with arbutin, MSH-α and UV-B irradiation). The results show that there was no significant difference in melanocyte proliferation and melanogenesis between arbutin and MSH-α treatment. However, the co-culture model was more stable than the pure culture model in terms of melanocyte proliferation and melanogenesis upon UV-B irradiation. Therefore, the co-culture model was superior to the pure culture as a useful method for the study of melanocytes and epidermal melanin unit.     

Toll‐like receptors 2 and 3 enhance melanogenesis and melanosome transport in human melanocytes

Because little is known about how the innate immune response influences skin pigmentation, we examined whether Toll‐like receptor (TLR) agonists participate in melanogenesis and melanosome transportation. We observed that TLR2/2 agonist HKLM and TLR3 agonist Poly(I:C) increased the amount of extracellular melanin from primary human epidermal melanocytes. HKLM, but not Poly(I:C), increased the melanogenic genes such as tyrosinase and dopachrome tautomerase. Poly(I:C) increased the expression of Rab27A, a molecule that facilitates melanosome transport to perimembranous actin filament. UVB irradiation induced Rab27A and melanosome transportation in a similar manner of Poly(I:C). SiRNA for TLR3 or Rab27A suppressed the perimembranous accumulation of Gp100‐positive vesicles in melanocytes and decreased melanin transfer to neighboring keratinocytes induced by both Poly(I:C) and UVB. These results suggest that the microenvironment in the epidermis and innate immune stimuli, such as microbiome and ultraviolet represented here by TLR2 and TLR3 agonists, could affect the melanogenesis in human melanocytes.     

Effect of streptomycin on melanogenesis and antioxidant status in melanocytes

Streptomycin is an aminoglycoside antibiotic with an antituberculosis activity commonly used in clinical practice due to its good antimicrobial characteristics. A well-known undesirable side effect of this drug is ototoxicity, which may be caused by overproduction of reactive oxygen species and loss of melanocytes in the inner ear. The aim of this study was to examine the effect of streptomycin on melanogenesis and antioxidant defense system in cultured normal human melanocytes (HEMa-LP). Streptomycin induced concentration-dependent loss in melanocytes viability. The value of EC₅₀ was determined to be ~5.0 mM. It has been shown that streptomycin causes inhibition of tyrosinase activity and reduces melanin content in human melanocytes in a concentration-dependent manner. Significant changes in the activity of cellular antioxidant enzymes: superoxide dismutase, catalase, and glutathione peroxidase were also stated. The results obtained in vitro may explain a potential role of melanocytes and melanin in the causative mechanisms of aminoglycosides ototoxic effects in vivo.     

A simple method for the establishment of tissue culture melanocytes from regenerating fowl feathers

In Vitro Cellular &; Developmental Biology - Plant - A quick and simple method for the establishment of tissue cultures of nonembryonic domestic fowl melanocytes was desired. The selected source...     

A ROCK inhibitor promotes keratinocyte survival and paracrine secretion,enhancing establishment of primary human melanocytes and melanocyte–keratinocyte co‐cultures

Primary melanocytes isolated from skin and expanded in culture have been widely used for laboratory research and clinical applications. The conventional method to isolate primary melanocytes from skin usually requires about 3–4 weeks of culture for melanocytes to grow sufficiently to passage. Considering that melanocytes comprise only 3%–7% of epidermal cells in normal human skin, it would be extremely helpful to increase the isolation efficiency and shorten the initial culture time to quickly meet various application needs. Here, we report that adding Y‐27632, a Rho kinase inhibitor, into the initial culture medium for 2 days can dramatically increase the yield of melanocytes. We found that Y‐27632 can promote keratinocyte attachment and survival in the melanocyte culture system, resulting in not only better recovery, but also increased proliferation of melanocytes by a paracrine signaling pathway. More specifically, Y‐27632 significantly induced keratinocyte expression of stem cell factor, which played an important role in enhancing the growth of melanocytes. In summary, Y‐27632 could profoundly enhance the yield of primary melanocytes in the initial culture through paracrine effects on keratinocytes.     

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.     

Characterization of melanin in human iridal and choroidal melanocytes from eyes with various colored irides

Variance in iris color is related to the incidence of several important ocular diseases, including uveal melanoma and age-related macular degeneration. The purposes of this study were to determine the quantity and the types of melanin in cultured human uveal melanocytes in relation to the iris color. Sixty-one cell cultures of pure uveal melanocytes were isolated from donor eyes with various iris colors. The amount of eumelanin (EM) and pheomelanin (PM) of these cells was measured by chemical degradation and microanalytical high-performance liquid chromatography (HPLC) methods. The total amount of melanin was measured by both microanalytical methods and spectrophotometry. Total melanin content, measured by HPLC and spectrophotometry, correlated well with r = 0.872 (P < 0.0001). The quantity and type of melanin in iridal and choroidal melanocytes showed no significant difference (P > 0.05). When cells became senescent, the levels of EM, PM and total melanin were significantly increased. In both growing and senescent melanocytes, the quantity and type of melanin were closely correlated to the iris color. In cells from eyes with dark-colored irides (dark brown and brown), the amount of EM, the ratio of EM/PM and total melanin were significantly greater than that from eyes with light-colored irides (hazel, green, yellow-brown and blue) (P < 0.0001). The quantity of PM in uveal melanocytes from eyes with light-colored irides was slightly greater than that from dark-colored irides, although not statistically significant (P > 0.05). The present study shows that iris color is determined by both the quantity and the type of melanin in uveal melanocytes. These results suggest a possibility that uveal melanin in eyes with dark-colored irides is eumelanic at the surface and acts as an antioxidant while that in eyes with light-colored irides exposes pheomelanic core and behaves as a pro-oxidant.