Effects Of Wnt3a Protein On The Melanogenesis Of Mouse Melanocyte Lineage

Background:Melanocytes synthesize melanin in melanosome and transfer the melanin granules tothe adjacent keratinocytes, where melanins are accumulated to generate pigmented skin orhairs. High melanin content protects the skin from harmful traviolet rays owning to theability to absorb UV radiation and quench the UV-induced intracellular free radicals.Defects in or a lack of melanocytes can lead to pigment disorders, such as piebaldism,albinism, vitiligo, and hair graying. Hair graying is one of the prototypical symbols ofhuman aging. Recent studies demonstrated that, the loss of hair pigmentation seen withhuman aging is caused by the loss of melanocytes and melanocyte stem cells.Repigmentation of vitiligo depends on available melanocytes. Recent studies found that,melanocyte stem cells located in the bulge region of the hair follicle are the most importantsources of functional melanocytes that can contribute to recovery in vitiligo.Melanocytes originate from neural crest-derived melanoblasts. During embryogenesis,melanoblasts migrate through the dermis into the epidermis. In the hairy region ofmammalian skin, melanoblasts further enter the newly developing hair follicles (HFs)where they finally become localized. Henceforward, melanoblasts are separated into twosubpopulations: differentiated melancytes, localized in the hair bulb, expresses MITF, DCT,TRP1, and tyrosinase, responsible for hair pigmentation; and melanocyte stem cells(McSCs), localized in the bulge region, expresses only DCT, responsible for therepopulation of the melanocyte system in subsequent hair cycles. In mature hair follicles,the melanocyte lineage consists of three distinct compartments: melanocyte stem cells,melanocyte progenitor cells (transient amplifying cells; TA cells) and terminallydifferentiated melanocytes (mature melanocytes).Melanocyte lineage in HFs repeatedly proliferate and differentiate during every haircycle. Melanocyte stem cells are quiescent during the rest phase (telogen) of hair cycle. With the initiation of the active growth (anagen) of hari cycle, melanocyte stem cellsproliferate, and give rise to melanocyte progenitor cells. These TA cells differentiate tomature melanocytes and produce melanin to the adjacent keratinocytes which growing hairbecomes pigmented. Melanocytes begin to shut down melanogenesis in late anagen andregression phase (catagen), and die by apoptosis from the hair bulb in rest phase (telogen),but reappear at the same location when HFs reenter anagen.Wnt3a has been reported to act through the canonical pathway and has important rolesin the development of melanocytes. Mutant mice deficient in Wnt1and Wnt3a are almostcompletely devoid of pigment cells. In vitro studies found that neural crest cells depleted ofWnt1and Wnt3a become neurons rather than melanocytes, while treatment with Wnt3acaused them to become pigment cells rather than neurons and glial cells. Wnt3a acts onmelanoblasts promote them differentiating to become melanocytes.Although the importance of Wnt3a in melanocyte development has been wellrecognized, the effect of Wnt3a in postnatal melanocytes has not been clearly elucidatedyet.Objective:To examine the presence and location of Wnt3a in mouse HF during hair cycle and toinvestigate the effects of Wnt3a on melanogenesis of the mouse melanocyte lineage (maturemelanocytes and immature melanocytes--melanocyte stem cells and TA cells), and toelucidate the possible mechanisms involved.Methods:1. Research on the expression pattern of Wnt3a signaling in hair follile melanocytelinage(1) By using Dct-LacZ transgenic mice, the protein expression pattern of Wnt3a wasobserved by X-gal staining and immunohistochemistry in the slides of skin in hair cycle.The mRNA and protein expression quantity of Wnt3a was observed by RT-PCR andWestern Blot in the whole skin in hair cycle.(2) The expression patterns of β-catenin and Lef-1in HF melanocyte lineage duringthe hair cycle were detected by X-gal staining and immunohistochemistry in the slides ofskin in hair cycle.2. Research on the effects of Wnt3a on the proliferation and melanogenesis of mouse melanocyte lineage in vitro(1) AdWnt3a, AdGFP, AdSimMITF were amplified in HEK293cells, and purified bycentrifuging in CsCl gradient.(2) Used "melan-a" as a mature melancoyte cell model, and infected melan-a cellswith AdWnt3a at a pre-determined optimal titer. The ability of AdWnt3a to activateWnt/β-catenin signaling was evaluated by Western blot and TOP/FOP-flash luciferasereporter assay. The effect of Wnt3a on melancoyte proliferation was evaluated by BrdUincorporation, cell countassay and cell cycle analysis. The effect of Wnt3a on melancoytemelanin synthesis was analyzed by melanin content and tyrosinase activity. The effect ofWnt3a on mRNA and protein levels of MITF, DCT, TRP1, and tyrosinase was analyzed byRT-PCR and Western Blot.(3) Used "iMC23" as an immature melancoyte cell model, to analyze the effect ofWnt3a on the melanogenesis of immature melancoytes, melanin production and tyrosinaseactivity assay were performed. The effect of Wnt3a on immature melancoyte differentiationwas analyzed by morphological observation and melanin synthesis.(4) Cultured primary mouse melanocytes as nomal melancoyte cell model, the effect ofWnt3a on the melanogenesis were analyzed by morphological observation and melaninproduction observation.3. Research on the effects of Wnt3a on the melanogenesis of mouse melanocytes invivoThe effect of Wnt3a on the melanogenesis of mouse melanocytes in vivo was evaluatedby intradermal administration of adenoviruses, and the statuses of hair follicle melanocyteswere assessed.Results and discussions:1. The expression pattern of Wnt3a signaling is temporally and spatially related withhair cycle.(1) Wnt3a was expressed in the epidermis, bulge, IRS precursors and hair bulb inanagen, decreased in catagen, and was basically not expressed in telogen. Somemelanocytes residing in the hair bulb also expressed Wnt3a in anagen.(2) Both β-catenin and Lef-1displayed nuclear positivity in hair bulb melanocytes inanagen HFs, suggesting that Wnt/β-catenin signaling was activated in HF melanocytes during anagen.2. Research on the effects of Wnt3a on the melanogenesis of mouse melanocytelineage in vitro(1) The adenoviruses (AdWnt3a, AdGFP and AdSimMITF) were propagated andpurified.(2) The effects of Wnt3a on the proliferation and melanogenesis of maturemelanocytes:AdWnt3a mediated efficient gene transfer and expressed Wnt3a protein in melan-acells. AdWnt3a-infected cells displayed higher ratios of TOP-flash/FOP-flash compared toAdGFP-infected cells demonstrated that AdWnt3a efficiently activated Wnt/β-cateninsignaling in melan-a cells.Wnt3a inhibited the proliferation of melan-a cells and this was associated withdecrease of cells in S phase and increase of cells in G1phase.Wnt3a significantly promoted melanogenesis of melan-a cells through upregulationthe expression of MITF and its downstream target genes, tyrosinase and TRP1.(3) The effects of Wnt3a on the melanogenesis of immature melanocytes: iMC23cellsexhibited characteristic small and bipolar cell bodies but, after treatment with AdWnt3a, thecell size and melanin synthesis increased noticeably, demonstrated that Wnt3a promotedimmature melanocytes differentiation and melanogenesis.(4) The effects of Wnt3a on the melanogenesis of primary mouse melanocytes: Wnt3apromoted primary melanocytes differentiation and melanin synthesis.3. Research on the effects of Wnt3a on the melanogenesis of mouse melanocytelineage in vivo.(1) Wnt3a promoted melanocyte stem cells differentiation.(2) Wnt3a promoted melanogenesis of HF melanocytes and it could rescue theexpression of MITF when silence endogenous MITF.Conclusion:We demonstrate that Wnt3a signaling is activated in mouse HF melanocytes duringanagen of hair cycle. In vitro, Wnt3a reduces the proliferation of mouse maturemelanocytes while simultaneously increasing the melanogenesis through upregulation ofMITF and its downstream genes. And Wnt3a also promotes the melanogenesis of mouse imature melanocytes. In vivo, Wnt3a rescued the expression of MITF when silenceendogenous MITF on HF melanocytes and promoted melanin synthesis. Our results suggestthat Wnt3a plays an important role in mouse HF melanocytes homeostasis.