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. Pre-treatment 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.
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. Pre-treatment 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.