| Background:Mast cells(MCs)are derived from bone marrow hematopoietic stem cells,which become mast cell progenitors after multiple differentiation in bone marrow.These progenitor cells circulate in the blood and migrate into peripheral tissues,where they further differentiate into mature MCs.MCs are endowed with a large repertoire of surface receptors allowing them to identify pathogens and to promote their clearance.Upon activation,MCs proliferate rapidly and migrate to the inflammatory site,then release inflammatory mediators that modulate the recruitment and activation of other leukocytes,ultimately regulating immune responses and involving in type I hypersensitivity reactions.Although the activation and proliferation of MCs are finely regulated by multiple signaling pathways,FcεRI-and C-Kit-mediated signaling pathways play an important role in MCs activation and proliferation.The IgE/Ag complex bind to FcεRI,triggering"LAT1/PLC-γ1/MAPKs" signaling pathway and intracellular calcium mobilization,promotes the degranulation of MCs and the release of inflammatory mediators,ultimately regulating type I hypersensitivity reactions.Upon binding with SCF,C-Kit undergoes dimerization and autophosphorylation,which activates " RAS-MAPKs" and"PI3K-AKT" signaling pathway.involved in the regulation of MCs proliferation,maturation and survival.FcεRI-and C-Kit-mediated signaling pathways are precisely regulated by a network of positive and negative regulators.The function abnormally of negative regulators can lead to persistent or excessive activation of FcεRI-and C-Kit-mediated signaling pathways,which eventually trigger the development of allergic diseases and mastocytosis.Discovery and exploration of precise negative regulators and the regulatory mechanisms that are involved in MCs activation and proliferation not only enrich the regulatory network of FcεRI-and C-Kit-mediated signaling pathways,but also provide new ideas for the effective diagnosis and treatment of allergic diseases and mastocytosis.Protein 4.1R was first identified in erythrocytes and had three highly conserved functional domains,including FERM,CTD and SAB,which performed different functions in red blood cells and nucleated cells.Our previous studies found that protein 4.1R negatively regulated CD4+T lymphocyte activation and proliferation by inhibiting TCR-mediated signal transduction.In a mouse model of experimental autoimmune encephalomyelitis,the 4.1R knockout mice were shown to be more sensitive to antigen with earlier and more severe onset.Recently,we found that protein 4.1R negatively regulated B cells activation and proliferation by inhibiting TLR4-mediated signal transduction.These results suggest that protein 4.1R may be a common negative regulator in the activation and proliferation of immune cells.MCs,as primary effector cells of type I hypersensitivity,are involved in the pathogenesis of asthma.The analysis of the GEO database revealed that 4.1R mRNA expression in the peripheral blood of asthmatic patients was significantly lower than that of 4.1R in the peripheral blood of normal subjects.In this study,we explored the role of protein 4.1R in MCs activation and proliferation.It layed a theoretical foundation for the diagnosis and treatment of diseases in which abnormal proliferation and activation of mast cells were involved.Methods:1.Structural analysis and prediction of protein 4.1RThe protein 4.1R from 8 species were searched by UniProt database,and multisequence alignment was carried out by Clustal W and ESPript.At the same time,MEGA and iTOL were used to construct the evolutionary tree.The physicochemical properties and secondary structure of human and mouse protein 4.1R were analyzed by ProtParam and PSIPRED,and the functional domain of protein 4.1R was analyzed by Pfam.2.The regulatory role of protein 4.1R in MCs activation was detected by transcriptome sequencing analysisBone marrow cells were harvested from the femurs and tibias of 4.1R+/+and 4.1R-/mice under aseptic conditions.After 4 weeks in culture,the purity of bone marrow mast cells(BMMCs)was detected by flow cytometry.After anti-DNP IgE and DNP-BSA complexes stimulation,the differentially expressed genes(DEGs)in 4.1R+/+and 4.1R/-BMMCs were detected by transcriptome sequencing.The DEGs were enriched by GO and KEGG enrichment analysis.Finally,a protein-protein interaction(PPI)network was constructed by STING and Cytoscape to screen the key nodal proteins.3.The effect of protein 4.1R on MCs activationAfter anti-DNP IgE and DNP-BSA complexes stimulation,the release of βhexosaminidase and histamine in 4.1R+/+and 4.1R-/-BMMCs were detected by the absorption photometry.The expression and secretion of cytokines(IL-6,IL-13 and TNF-α)and chemokines(CCL-2)were analyzed by qRT-PCR and ELISA.The asthmatic model was induced by OVA in 4,1R+/+and 4.1R-/-mice.The number of inflammatory cells in bronchoalveolar lavage fluid,inflammatory cell infiltration and hyperplasia of mucus-secreting goblet cells in lung tissues were statistically analyzed.The expression of IL-4,IL-5 and IL-13 in lung tissues were measured by qRT-PCR.To further explore the mechanism of 4.1R regulating BMMCs activation,the phosphorylation levels of Lyn,Syk,LAT1,PLCγ1,ERK1/2,JNK1/2 and p38 in 4.1R+/+and 4.1R-/-BMMCs were detected by western blotting(WB).The calcium flux of 4.1R+/+and 4.1R-/-BMMCs were measured by flow cytometry.The interaction between 4.1R and LAT1 was detected by immunofluorescence(IF)and co-immunoprecipitation(CO-IP).4.The effect of protein 4.1R on MCs proliferationThe gene of EPB41 in P815 cells was knocked out by CRISPR/Cas9 and the expression of 4.1R in P815-4.1R-/-cells was confirmed by WB.The proliferation,apoptosis and cell cycle of P815-4.1R+/+and P815-4.1R-/-cells were detected by colony formation and flow cytometry.P815-4.1R+/+ and P815-4.1R-/-cells were injected in retro-orbital sinus of DBA/2 mice to establish aggressive systemic mastocytosis(ASM)model.The survival time of model mice was observed and the swelling degree of liver and spleen was analyzed.The phosphorylation levels of C-Kit,Lyn,ERK1/2,JNK1/2 and AKT in P815-4.1R+/+and P815-4.1R-/-cells were measured by WB.The interaction between 4.1R and C-Kit was measured by CO-IP.Results:1.Mouse and human 4.1R proteins are evolutionarily conserved and structurally similarThrough multiple sequence alignment and evolutionary tree analysis,we found that the homology between mouse and human protein 4.1R was 90.42%,and the evolutionary distance was close.The results of physicochemical properties and structure analysis showed that the physical and chemical properties,secondary structure and functional domain of human and mouse protein 4.1R were highly similar.These results suggestted that mouse and human 4.1R might have similar functions.The mice could be used to explore the role of 4.1 R in MCs activation and proliferation.2.Transcriptome sequencing analysis indicated that protein 4.1R regulates mast cell activation through the FcεRI signaling pathway4.1R+/+and 4.1 R-/-BMMCs with purity>90%can be used in subsequent experiments.After anti-DNP IgE and DNP-BSA complexes stimulation,768 DEG were identified between 4.1R-/-BMMCs and 4.1R+/+BMMCs.The GO and KEGG enrichment analysis showed that deletion of 4.1R affected MCs activation and FcεR1 signaling pathway.The results of PPI showed that 4.1R might be closely related to cytokines(IL-6,IL-13,TNF-α)and chemokines(CCL-2).These results suggested that protein 4.1R might inhibit MCs activation by regulating FcεRI-mediated signaling pathway.3.Protein 4.1R inhibits MCs activation by negatively regulating the FcsRI signaling pathway4.1R+/+and 4.1R-/-BMMCs with purity>90%can be used in subsequent experiments.The release rate of β-hexosaminidase and histamine from 4.1R-/-BMMCs are significantly increased compared to the 4.1R+/+BMMCs.The expression levels of IL6,IL-13,TNF-α and CCL2 were markedly higher in 4.1R-/-BMMCs than that in 4.1R+/+BMMCs.Compared with the OVA sensitized 4.1R+/+ mice,the number of inflammatory cells in bronchoalveolar lavage fluid,inflammatory cell infiltration and hyperplasia of mucus-secreting goblet cells in lung tissues significantly increased in OVA sensitized 4.1R-/-mice.Moreover,the mRNA expression levels of IL-4,IL-5 and IL-13 were significantly upregulated in the lung tissues from OVA sensitized 4.1R-/-mice.Compared with the 4.1R+/+BMMCs,the phosphorylation of the FcεRI-proximal tyrosine kinases(Lyn and Syk),adaptor protein(LAT1)and phospholipase(PLC-γl)were significantly increased in the 4.1R-/-BMMCs.FcεRI-mediated phosphorylation of the MAPKs,such as ERK1/2 and JNK1/2,were also significantly enhanced in the absence of 4.1R.Meanwhile,4.1R deficiency resulted in a consistently sustained calcium concentration in the cytoplasm of BMMCs.IF and CO-IP results showed that protein 4.1R could interact with LAT1 to regulate FcεRI-mediated signaling pathway.These results suggested that protien 4.1R,affecting the activation of LAT1,inhibited FcεRI-mediated signal transduction to negatively regulate the activation of MCs,ultimately exacerbated allergic asthma.4.Protein 4.1R inhibits MCs proliferation by negatively regulating the C-Kit signaling pathwayWe firstly constructed a 4.1R gene knock out P815 mastocytoma cells by using CRISPR/Cas9 technique.Lack of 4.1R significantly enhanced P815 cells proliferation and colony formation.Compared with P815-4.1R-/+cells,the percentage of P815-4.1R/-cells in the G0-G1 phase significantly reduced and the percentage of cells in the S and G2-M phase markedly increased.The apoptosis rate of P815-4.1 R+/+and P815-4.1 R-/cells were comparable.Retro-orbital injections of P815-4.1R-/-cells in DBA/2 mice resulted in rapid development of ASM.Compared with the P815-4.1R+/+-DBA/2 mice,the weight of the spleen and liver in P815-4.1R-/--DBA/2 mice significantly increased.Moreover,4.1R deficiency resulted in a striking enhancement in the phosphorylation of Lyn,ERK1/2,JNK1/2 and AKT,which were crucial for P815 cells proliferation.WB and CO-IP results showed that 4.1R could interact with C-Kit to inhibit C-Kit phosphorylation.These results suggested that protein 4.1R,affecting the activation of C-Kit,inhibited C-Kit-mediated signal transduction to negatively regulate the proliferation of MCs,ultimately exacerbated mastocytosis.Conclusions:Taken together,our findings demonstrate that protein 4.1R negatively regulates MCs activation and proliferation by inhibiting FcεRI and C-Kit mediated signal transduction.It may provide a new idea for the prevention and treatment of allergic asthma and mastocytosis. |
