Abrogating effect of N-linked carbohydrate modifiers on the stem cell factor and endothelin-1-stimulated epidermal pigmentation in human epidermal equivalents

Background: We previously demonstrated that the hyperpigmentation that occurs in UVB-melanosis as well as in solar lentigos is associated with the increased production of melanogenic cytokines, such as endothelin (EDN)-1 and stem cell factor (SCF), by keratinocytes in those areas of the skin. Objective: We developed a model for these hyperpigmentary disorders in EDN1 + SCF stimulated human epidermal equivalents (HEEs) and characterized the effects of the N-linked carbohydrate core synthesis inhibitor glucosamine or N-linked carbohydrate processing inhibitors deoxynojirimycin or monensin on the stimulated HEE pigmentation. Methods: Those effects were assessed by melanin analysis, real-time RT-PCR and Western blotting. Results: The addition of these N-linked carbohydrate modifiers (NCMs) markedly abolished the EDN1 + SCF-elicited increase in HEE pigmentation over 14 days. Real-time RT-PCR and Western blotting of these NCM-treated HEEs unexpectedly revealed that the EDN1 + SCF-stimulated steady-state levels of tyrosinase (TYR), TYR-related protein-1, dopachrome tautomerase and PMEL17 as well as microphthalmia-associated transcription factor (MITF) were significantly attenuated at the transcriptional and translational levels without any cytotoxic effects on keratinocytes and melanocytes in the HEEs. Pretreatment of cultured normal human melanocytes with the NCMs interrupted the EDN1 + SCF-induced stimulation of steady-state levels of MITF at the transcriptional and translational levels and TYR activity without any direct inhibitory effect on the catalytic activity of TYR in vitro. Conclusion: This study provides evidence that NCMs have a potential to attenuate the EDN1 + SCF-stimulated pigmentation of HEEs by abrogating the increased steady-state levels of MITF mRNA, which results in the attenuation of the increased steady-state levels of these melanocyte-specific proteins. (C) 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.