摘要
Melanin, synthesized by melanocyte, is transferred to neighboring keratinocyte and finally accumulates in perinuclear site. Except functioning as an internal sunscreen to protect from UV damage, the potential effect of melanin on modulating the bioactivity of keratinocyte has not yet been fully investigated. In this study, we added melanin directly to the culture of human epidermal keratinocytes and the uptake of melanin was found to be dose- and time-dependent as determined by spectrophotometric method. The uptaken melanin accumulated perinuclearly in keratinocytes that is similar to the pattern observed in human solar lentigo tissue by microscopic examination. Pretreatment of keratinocytes with either niacinamide or trypsin inhibitor reduced the uptake of melanin dose-dependently, indicating a PAR-2-dependent pathway involved. Melanin uptake by keratinocytes inhibited cell proliferation as demonstrated both by the decrease of cell number and nuclear Ki-67 expression. Inhibited Ki-67 expression in melanin-containing keratinocyte was also found in human lentigo tissue. The cell cycle arrested at G1 phase in melanin-uptaken keratinocytes was confirmed by flow cytometric method. The protein expressions of cyclin-dependent kinase 1 (CDK1), CDK2, cyclin E, cyclin A and cyclin B were significantly reduced by melanin treatment. Microarray analysis, RT/real-time PCR and western blot demonstrated the inhibited expression of DKK1, a protein known to reduce skin pigmentation, in melanin-uptaken keratinocytes. Together, the direct incubation of keratinocyte with melanin might serve as a useful model to study the potential mechanisms involved in melanin uptake and pigmentation process.
Melanin, synthesized by melanocyte, is transferred to neighboring keratinocyte and finally accumulates in perinuclear site. Except functioning as an internal sunscreen to protect from UV damage, the potential effect of melanin on modulating the bioactivity of keratinocyte has not yet been fully investigated. In this study, we added melanin directly to the culture of human epidermal keratinocytes and the uptake of melanin was found to be dose- and time-dependent as determined by spectrophotometric method. The uptaken melanin accumulated perinuclearly in keratinocytes that is similar to the pattern observed in human solar lentigo tissue by microscopic examination. Pretreatment of keratinocytes with either niacinamide or trypsin inhibitor reduced the uptake of melanin dose-dependently, indicating a PAR-2-dependent pathway involved. Melanin uptake by keratinocytes inhibited cell proliferation as demonstrated both by the decrease of cell number and nuclear Ki-67 expression. Inhibited Ki-67 expression in melanin-containing keratinocyte was also found in human lentigo tissue. The cell cycle arrested at G1 phase in melanin-uptaken keratinocytes was confirmed by flow cytometric method. The protein expressions of cyclin-dependent kinase 1 (CDK1), CDK2, cyclin E, cyclin A and cyclin B were significantly reduced by melanin treatment. Microarray analysis, RT/real-time PCR and western blot demonstrated the inhibited expression of DKK1, a protein known to reduce skin pigmentation, in melanin-uptaken keratinocytes. Together, the direct incubation of keratinocyte with melanin might serve as a useful model to study the potential mechanisms involved in melanin uptake and pigmentation process.
