The effect of the NMDA receptor-dependent signaling pathway on cell morphology and melanosome transfer in melanocytes

Background: The pigmentation of skin and hair in mammals is driven by the intercellular transfer of melanosome from the melanocyte to surrounding keratinocytes However, the detailed molecular mechanism is still a subject of investigation. Objective: To investigate the effects of N-methyl-D-aspartate (NMDA) receptor-dependent signaling pathway on melanocyte morphologic change and melanosome transfer between melanocytes and keratinocytes. Methods: The expression and the intracellular distribution of NMDA receptor in human melanocyte were analyzed by Western blot and immunofluorescence staining. Melanocytes were treated with 100 mu M NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleatel and 100 mu M NMDA receptor agonist NMDA, after which the morphological change of melanocyte dendrites and filopodias were observed by scanning electron microscope. The beta-tubulin distribution and intracellular calcium concentration ([Ca2+](i)) were observed by immunofluorescence staining and flow cytometry under the same treatment respectively. In addition, melanocytes and keratinocytes were co-cultured with or without treatment of MK-801, and the melanosome transfer efficacy were analyzed by flow cytometry. Results: We show that human epidermal melanocytes expresses NMDA receptor 1, one subtype of the ionotropic glutamate receptors (iGluRs). Stimulation with agonist of NMDA receptor increased the number of melanocyte filopodia. In contrast, blockage of NMDA receptor with antagonist decreased the number of melanocyte filopodia and this morphological change was accompanied by the disorganization of beta-tubulin microfilaments in the intracellular cytoskeleton. In melanocyte-keratinocyte co-cultures, numerous melanocyte filopodia connect to keratinocyte plasma membranes; agonist of NMDA receptor exhibited an increased number of melanocyte filopodia attachments to keratinocyte, while antagonist of NMDA receptor led to a decreased. Moreover, antagonist of NMDA receptor decreased the intracellular calcium concentration in melanocytes and reduced the efficacy of melanosome transfer. Conclusion: Our data suggest that filopodia delivery is the major mode of melanosome transfer between melanocytes and keratinocytes. NMDA drives melanosome transfer by promoting filopodia delivery and direct morphological effects on melanocytes, while MK-801 affects the intracellular beta-tubulin redistribution and the filopodia delivery between melanocytes and keratinocytes. We hypothesize that NMDA receptor-dependent signaling is involved in melanosome transfer, which is associated with calcium influx, cytoskeleton protein redistribution, dendrites and filopodia formation. A thorough understanding of melanosome transfer is crucial for designing treatments for hyper- and hypo pigmentary disorders of the skin. (C) 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.