Receptor-interacting Protein 1 Is A Key Mediator In Oxidative Stress-induced Necroptosis Of Melanocytes

Backgound:Vitiligo is an acquired,depigmenting disorder of the skin,which affects about 0.5%to2%of the world population,and gives patients a severe mentally burden.There is still a lack of effective treatment for vitiligo because of the unknown pathogenesis.Researches show that oxidative stress plays a pivotal role in occurrence and progression of vitiligo.We and others found that oxidative stress produced a great deal of oxygen free radicals including H₂O₂,which induced apoptosis and dysimmunity in melanocytes,and then formed cascade reaction further damaging melanocytes.Recent studies showed that oxidative stress also induced necroptosis,a kind of nonapoptotic,yet regulated cell death,besides apoptosis.There is no existing evidence whether or not oxidative stress could induce necroptosis in melanocytes.Receptor-interacting protein 1（RIP1）,also named RIPK1,is a member of RIP family,which is discovered in tumor necrosis factor（TNF）signaling pathway.When bonding to TNF,TNF receptor interacts with TRADD,and then recruit RIP1 and TRAF2,IAPs and LUBAC complex,to form complex I,which leads to activation of mitogen-activated protein kinases（MAPKs）or nuclear factor kappa-light chain-enhancer of activated B cells（NF-κB）.Once cellular conditions change,RIP1,modified posttranslationally,interacts with FADD and caspase-8 to form complex IIa,which induces apoptotic cell death.When caspase activity is inhibited,interaction of RIP1 with RIP3,namely necrosome,triggers phosphorylation of RIP3,which phosphorylates mixed lineage kinase domain like pseudokinase（MLKL）.Once phosphorylation,MLKL translocates to plasm membrane and ruptures it,which is the process of necroptosis.What the above indicates is that RIP1is essential in cell survival and death.A recent research showed that oxidative stress induced by high concentration of Tert-butyl hydroperoxide（t-BHP）in endothelial cells caused necrosome formation and necroptosis.When silencing RIP1,RIP3 and MLKL in endothelial cells,or pretreating cells with RIP1 inhibitor necrostatin-1（Nec-1）and MLKL inhibitor necrosulfonamide（NSA）,cell death induced by t-BHP decreased.Moreover,RIP1 knockout in mouse embryonic fibroblasts（MEF）and human leukemia Jurkat cells reduced their sensitivity to H₂O₂–induced cell death.Therefore,we hypothesized that RIP1 is of vital importance in H₂O₂-induced oxidative damage in melanocytes,and hypothesized that vitiligo melanocytes were vulnerable to H₂O₂-mediated oxidative stress probably due to upregulation of RIP1,RIP3 and MLKL,or necrosome formation more and earlier.Aims:1.To explore the expression of RIP1 and its necroptotic signaling molecules,and to figure out the role of RIP1 in melanocytes under oxidative stress condition.2.To investigate the underlying mechanism of RIP1 and its necroptotic signaling molecules involved in oxidative stress-induced melanocytes damage.Methods:1.Firstly,we detected expression of p-RIP3 and p-MLKL in melanocytes of vitiligo lesional skin by confocal microscopy to testify whether or not necroptosis exists in vitiligo melanocytes.2.Secondly,we treated human immortalized normal melanocytes（PIG1）and vitiligo melanocytes（PIG3V）with 1.0 mM H₂O₂ to measure cell viability and Propidium Iodide positive（PI⁺）rate by CCK-8 assay and flow cytometry（FCM）respectively.Meanwhile,mRNA and protein level of RIP were detected by Real-time PCR and Western blot.3.We detected cell viability and PI⁺rate under H₂O₂ stimulation after knocking-down RIP1 by transfection with lentivirus-shRNA against RIP1（LV-RIP1-shRNA）,silencing MLKL by siRNA,pretreating with Nec-1 and NSA respectively in PIG1 and PIG3V.4.After treating cells with H₂O₂,we focused on RIP1 and RIP3 co-location and p-MLKL translocation to plasm membrane in PIG1 and PIG3V using immunocytofluorescense assay.5.To verify the phenomenon found in cell immunofluorescence,we detected interaction between RIP1 and RIP3 in PIG1 and PIG3V by immunoprecipitation assay.6.Moreover,we monitored mitochondrial reactive oxygen species（mito-ROS）in PIG1 and PIG3V transfection with LV-RIP1-shRNA or pretreating with Nec-1 under H₂O₂ treatment.Then,after pretreating PIG1 and PIG3V with mito-ROS specific scavenger mito-TEMPO,we stimulated cells with H₂O₂ and measured cell viability.Besides,we detected interaction between RIP1 and RIP3 in PIG1 and PIG3V by immunoprecipitation assay under H₂O₂ stimulation with pretreatment of mito-TEMPO.7.We focused on the activation of MAPKs in PIG1 and PIG3V under oxidative stress when knocking-down RIP1 or pretreating with Nec-1.Results:1.Histoimmunofluorescence indicated that p-RIP3 and p-MLKL were detectable in melanocytes of vitiligo lesional skin,whereas neither of them existed in normal melanocytes.2.We treated PIG1 and PIG3V with 1.0 mM H₂O₂ for 24h and found that PI⁺cells increased significantly.It was noteworthy that PI⁺cells in PIG3V were more than those in PIG1.What’s more,mRNA and protein level of RIP1 in both PIG1 and PIG3V were upregulated in a time-dependent manner.3.Knocking-down RIP1 and pretreating Nec-1 increased cell viability and decreased PI⁺cells under H₂O₂ treatment.Meanwhile,both silencing MLKL in PIG1 and PIG3V and MLKL inhibitor NSA enhanced resistance to oxidative damage.4.Confocal microscopy showed that RIP1 and RIP3 interaction was enforced after stimulating with H₂O₂ for 6h,and p-MLKL translocated to plasm membrane after treating with H₂O₂ for 6h in PIG3V,which happened earlier than those in PIG1.5.We performed immunoprecipitation assay and found that the level of RIP3interacted with RIP1 was increased in a time-dependent manner under H₂O₂ stimulation,which was inhibited by Nec-1 pretreating.6.Mito-ROS was reduced both in PIG1 and PIG3V with RIP1 k nocking down and pretreating with Nec-1 after cells were exposed to H₂O₂.Mito-TEMPO decreased sensitivity to oxidative stress injury in PIG1 and PIG3V followed by H₂O₂ stimulation for12h.However,indistinguishable cell viability was noticed after PIG1 a nd PIG3V were exposed to H₂O₂ for 24h with miti-TEMPO pretreatment.Immunoprecipitation assay indicated that miti-TEMPO pretreatment restrained RIP1 interaction with RIP3 after PIG1and PIG3V were stimulated by H₂O₂ for 12h,which had no influence on the interaction when melanocytes were exposed to H₂O₂ for 24h.7.Neither knocking-down RIP1 nor Nec-1 pretreatment had a visible effect on MAPKs activation in PIG1 and PIG3V under H₂O₂ exposion.Conclusion:Oxidative stress induces necrosome formation in melanocytes,which phosphorylates MLKL,and then p-MLKL translocates to plasm membrane,damaging cell membrane integrity and causing cell death.PIG3V is vulnerable to oxidative stress comparing with PIG1,probably as necrosome formation and p-MLKL translocation to plasm membrane is faster.Additionally,elevation of mitochondrial ROS induced by oxidative stress further damages cell viability.More importantly,p-RIP3 and p-MLKL are detectable in biopsies of epidermal melanocytes in vitiligo patients.All in all,we came up with a new idea that necroptosis was a new-found choice of cell death induced by oxidative stress in melanocytes,which provided a novel pathogenesis in vitiligo.