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 100μM NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate] and 100μM NMDA receptor agonist NMDA, after which the morphological change of melanocyte dendrites and filopodias were observed by scanning electron microscope. The β-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 β-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.


CONCLUSIONS
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 β-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.
Dermatology Department of 1st Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China.
Jing
J
Ni
Dermatology Department of 1st Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China.
Nan
N
Wang
Dermatology Department of 1st Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China.
Lili
L
Gao
Dermatology Department of 1st Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China.
Lili
L
Li
Dermatology Department of 1st Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China.
Siwen
S
Zheng
Dermatology Department of 1st Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China.
Yuejian
Y
Liu
Dermatology Department of 1st Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China.
Molu
M
Ozukum
Dermatology Department of 1st Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China.
Anna
A
Nikiforova
Dermatology Department of 1st Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China.
Guangming
G
Zhao
Dermatology Department of 1st Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China. Electronic address: szqdalian@163.com.
Zhiqi
Z
Song
D018920:Coculture Techniques; D003599:Cytoskeleton / Q000378:metabolism; D016291:Dizocilpine Maleate / Q000737:chemistry; D004817:Epidermis / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000166:cytology; D008543:Melanins / Q000378:metabolism; D008544:Melanocytes / Q000166:cytology*; D020460:Melanosomes / Q000378:metabolism*; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010858:Pigmentation; D011554:Pseudopodia / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D015398:Signal Transduction; D012871:Skin Diseases / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
D016291:Dizocilpine Maleate; D008543:Melanins; D009419:Nerve Tissue Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D014404:Tubulin; C485668:GRIN1 protein, human
Calcium influx; Dendrite; Filopodia; Melanosome transfer; NMDA receptor