Post-translational processing of melanogenesis subsequent to melanogenic protein expression and its regulation leading to the development of novel depigmenting agents

Hyperpigmentation mechanisms including UVB-melanosis have been recently progressed in terms of paracrine cytokine regulation between melanocytes and keratinocytes/fibroblasts as well as of endothelins or stem cell factor-induced intracellular signalings leading to gene expression of melanogenic molecules including tyrosinase, endothelin B receptor or melanosomal protein Pmel17. According to these mechanisms, several novel depigmenting agents have recently been developed. However, post-translational processing ofmelanogenesis subsequent to gene and protein expression of melanogenic molecules including tyrosinase and Pmel 17 is poorly understood and there is few depigmenting agents developed based on the above strategy.Previously we found that the recovery process of melanization following glycosylation inhibition of B-16 melanoma cells which induces complete loss of melanization in melanosome due to the interrupted transfer of tyrosinases compartmentalized in coated vesicles is a good tool for searching post-translational regulation of melanogenesis. In this study, using the pigmentation recovery process of glycosylation-inhibited B-16 melanoma cells and western blotting analysis, we determined whether tyrosinases are originally present in immature melanosome or is transferred from Golgi area to melanosomes. In glycosylation-inhibited B-16 melanoma cells, tyrosinases are not localized in melanosome-rich fraction but in cytosol fraction, indicatingthat tyrosinases are transferred from Gogi area to melanosomes. The addition of reduced glutathione into the recovery process abolished the recovered translocation of tyrosinases from Gogi-endoplasmic reticulum-lysosome(GERL) to melanosomes despite no glycosylation inhibition potential of reduced glutathione, resulting in the continuous complete loss of melanization. These findings suggest that reduced glutathione serves as a translocation inhibitor for tyrosinases but not as a tyrosinase inhibitor. To assess whether glycosylation inhibitors have a potential to inhibit epidermal hyperpigmentation stimulated by melanogenic cytokines released from UVB-exposed keratinocytes, we used guinea pig skin organ culture or three-dimensional cultures consisting of human keratinocytes and human melanocytes to examine the inhibitory effect of glycosylation inhibitors on epidermal pigmentation. In both models, glycosylationinhibitors markedly abolished visibly increased pigmentation without affecting increased melanocyte population. The present study suggests that glycosylation inhibitors or reduced gluthathione can serve as novel depigmenting agents with unique strategy such as intracellular translocation inhibition if they can penetrate into the epidermis at effective concentrations when topically applied.