Ni Jing, Wang Nan, Gao Lili, Li Lili, Zheng Siwen, Liu Yuejian, Ozukum Molu, Nikiforova Anna, Zhao Guangming, Song Zhiqi
Dermatology Department of 1st Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China.
Dermatology Department of 1st Affiliated Hospital, Dalian Medical University, Dalian, Liaoning, China.
J Dermatol Sci. 2016 Dec;84(3):296-304. doi: 10.1016/j.jdermsci.2016.08.534. Epub 2016 Aug 24.
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.
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.
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 ([Ca]) 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.
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.
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.
哺乳动物皮肤和毛发的色素沉着是由黑素小体从黑素细胞向周围角质形成细胞的细胞间转移驱动的。然而，详细的分子机制仍是一个研究课题。
研究N-甲基-D-天冬氨酸（NMDA）受体依赖性信号通路对黑素细胞形态变化以及黑素细胞与角质形成细胞之间黑素小体转移的影响。
采用蛋白质免疫印迹法和免疫荧光染色法分析人黑素细胞中NMDA受体的表达及细胞内分布。用100μM NMDA受体拮抗剂MK-801[（+）-5-甲基-10,11-二氢-5H-二苯并[a,d]环庚烯-5,10-亚胺马来酸盐]和100μM NMDA受体激动剂NMDA处理黑素细胞，之后通过扫描电子显微镜观察黑素细胞树突和丝状伪足的形态变化。在相同处理下，分别采用免疫荧光染色法和流式细胞术观察β-微管蛋白分布及细胞内钙浓度（[Ca]）。此外，将黑素细胞和角质形成细胞进行共培养，有无MK-801处理，通过流式细胞术分析黑素小体转移效率。
我们发现人表皮黑素细胞表达离子型谷氨酸受体（iGluRs）的一种亚型——NMDA受体1。用NMDA受体激动剂刺激可增加黑素细胞丝状伪足的数量。相反，用拮抗剂阻断NMDA受体可减少黑素细胞丝状伪足的数量，这种形态变化伴随着细胞内细胞骨架中β-微管蛋白微丝的紊乱。在黑素细胞-角质形成细胞共培养体系中，众多黑素细胞丝状伪足与角质形成细胞质膜相连；NMDA受体激动剂使黑素细胞丝状伪足与角质形成细胞的附着数量增加，而NMDA受体拮抗剂则导致附着数量减少。此外，NMDA受体拮抗剂降低了黑素细胞内的钙浓度并降低了黑素小体转移效率。
我们的数据表明，丝状伪足传递是黑素细胞与角质形成细胞之间黑素小体转移的主要方式。NMDA通过促进丝状伪足传递以及对黑素细胞的直接形态学作用来驱动黑素小体转移，而MK-801影响细胞内β-微管蛋白的重新分布以及黑素细胞与角质形成细胞之间的丝状伪足传递。我们推测NMDA受体依赖性信号传导参与黑素小体转移，这与钙内流、细胞骨架蛋白重新分布、树突和丝状伪足形成有关。深入了解黑素小体转移对于设计皮肤色素沉着过多和过少疾病的治疗方法至关重要。
