The Regulating Role Of PP2Ac Methylation In M1 Macrophage Polarization

Objective:Protein phosphatase 2A(PP2A),a major serine/threonine protein phosphatase in eukaryotic cells.PP2A complex is a heterotrimer composed of an active core dimer that can exit independently,C subunit(PP2Ac)acts as the catalytic subunit of PP2A,is responsible for the dephosphorylation events.Leucine carboxy methyltransferase 1(LCMT1)and protein methylesterase(PPME-1)regulate the methylation/demethylation of leucine 309th site respectively and co-regulate the biological function of PP2A.however,the effect of PP2Ac methylation on the differentiation of M1 macrophages has not yet been reported.Mitochondria are the main organelles of autophagy in cells,which maintain the balance of glucose and lipid metabolism by clearing damaged mitochondria,and participate in cell proliferation,differentiation and the occurrence and development of diseases.In this study,we constructed a model of Ml macrophage polarization in vitro.ABL127 inhibited the activity of protein methylesterase-1(PPME-1)to increase the level of PP2Ac methylation,and LCMT1 siRNA was used to reduce the level of PP2Ac methylation.To investigate the effect of PP2Ac methylation modification on the polarization of M1 macrophage by mitophagy and glycolysis.Methods:1.The effect of PP2Ac methylation on Ml macrophage polarization1.1 Changes of PP2Ac methylation in Ml macrophage polarization1)THP-1 cells were treated with PMA for 24h to differentiate into M0 macrophages,and then stimulated with lipopolysaccharide(LPS)and interferon-?(OFN-?)for 48h to differentiate into M1 macrophages.2)Inverted microscope was used to observe the morphology changes of macrophages.3)The mRNA expression levels of cyclooxygenase 2(COX-2),tumor necrosis factor a(TNF-?),interleukin 6(IL-6)and C-X-C motif chemokine ligand 10(CXCL-10)in M1 macrophages were detected by quantitative real-time PCR.4)The expression of PP2Ac methylation in Ml macrophage polarization was detected by Western blot.1.2 The effect of PP2Ac methylation in M1 macrophage polarization1)The effect of ABL127 and LCMT1 siRNA on the mRNA expression of leucine carboxy methyltransferase 1(LCMT1)in Ml macrophages was detected by real-time fluorescence quantitative PCR.2)Western blot was used to detect the effect of ABL127 and LCMT1 siRNA on the expression of methylated-associated proteins in M1 macrophages.3)The effect of ABL127 and LCMT1 siRNA on M1 macrophage morpho-logy was observed by inverted microscope.4)qPCR was used to detect the effect of ABL127 and LCMT1 siRNA on the expression of M1 macrophages gene markers.1.3 The effect of ABL127 and LCMT1 siRNA on phagocytosis of Ml macrophage was detected by neutral red uptake assay2.The role of PP2Ac methylation in mitophagy of M1 macrophages1)The changes of lysosome(autophagy-lysosome)and mitochondria during the polarization of Ml macrophages were tested by fluorescence microscopy and fluorescence microplate reader.Detection of LC3,Beclin-1,p62 and PINK1 expression by Western blot to monitor the autophagy flux.2)The effects of ABL127 and LCMT1 siRNA intervention on mitochondrial membrane potential and ROS of M1 macrophages polarization were detected by fluorescence microscope and fluorescence microplate reader.3)Western blot was used to detect the expression of autophagy/mitophagy autophagy-associated protein Bcelin-1,p62,LC3,PINK1,Parkin,VDAC1 and NLRP3 after ABL127 and LCMTl siRNA intervention.3.The effect of PP2Ac methylation on Ml macrophage polarization via glycolysis pathwayDetermination of activities of glycolytic enzymes hexokinase(HK),pyruvate kinase(PK)and lactate dehydrogenase(LDH)in M1 macrophages by spectrophotometry.Results:1.The role of PP2Ac methylation on Mlmacrophage polarization1.1 The Changes of PP2Ac methylation in M1 macrophage polarization1)MO macrophages differentiated into M1 macrophages,cells were elong-ated and spindle-shaped.2)In the polarization of MO into Ml macrophages,the expression of related gene markers including cyclooxygenase 2(COX-2),tumor necrosis factor a(TNF-?),interleukin 6(IL-6)and chemotactic factor 10(CXCL-10)were significantly increased(p<0.01).3)MO macrophage polarized to M1 macrophages,the expression of PPME-1 and PP2Ac demethylation were decreased and the expression of LCMT1 and PP2Ac methylation were increased.1.2 The effect of PP2Ac methylation on Ml macrophage polarization1)Compared with the solvent control group,after treated with ABL127,the gene expression of LCMT1 was significantly higher in M1 macrophage(p<0.05).There was no significant difference in the expression level of total PP2Ac and PPME-1(p>0.05).The demethylation of PP2Ac was decreased,while the methylation of PP2Ac increased,the expression of LCMT1 gene and protein were increased.Compared with the control siRNA M1 macrophage,the gene expression of LCMT1 was significantly decreased after LCMT1 siRNA treatment(p<0.05),there was no significant difference in the protein expression of total PP2Ac and PPME-1,while the methylation of PP2Ac and LCMT1 were markedly reduced(p<0.05).2)Compared with the solvent control group,ABL127 intervention could significantly promote the expansion and outspread of M1 macrophages and increase the number of spindle-like cells.Compared with the control siRNA M1 macrophage,LCMT1 siRNA intervention could significantly reduce the number of spindle-shaped cells in M1 macrophages.3)Compared with the solvent control group,the gene expression of COX-2,TNF-? and IL-6,CXCL-10 were further increased after ABL127 intervention(p<0.05).Compared with the control siRNA Ml macrophage,the gene expression of COX-2,TNF-? and IL-6,CXCL-10 were further decreased after LCMT1 siRNA intervention(P<0.05).1.3 The effects of PP2Ac methylation on phagocytosis of Ml macroph-agesThe phagocytosis of M1 macrophages was significantly higher than that MO macrophages(p<-0.05).After treated with ABL127,the phagocytosis was further enhanced than the solvent control group(p<0.05).Compared with the negative control siRNA M1 macrophage,the phagocytosis was significantly decreased after LCMT1 siRNA intervention(p<0.05).2.The effect of PP2Ac methylation in mitophagy of M1 macrophage polarization1)In the polarization of MO into Ml macrophages,Lysosome/autolysosome were gradually increased with the prolongation of differentiation time,while mitochondria were decreased(p<0.05).During the differentiation of M1 macrophages,the expression of Beclin-1 and LC3,PINK1 were increased,while P62 decreased(p<0.05).Compared with solvent control group,ABL127 treatment could increase the number of lysosomes and decrease the number of mitochondria(p<0.05).Compared with the control siRNA M1 macrophage,the number of lysosomes was decreased and mitochondria was increased after LCMT1 siRNA intervention(p<0.05).2)MO macrophages differentiated into M1 macrophages,mitochondrial membrane potential was decreased and intracellular ROS was increased(p<0.05).Compared with solvent control group,ABL 127 could protect against mitochondrial membrane potential and decrease intracellular ROS.Compared with the control siRNA M1 macrophage,the membrane potential was further decreased and ROS was increased while LCMT1 siRNA intervention(p<0.05).3)The proteins expression of PP2Ac methylation,Beclin-1,LC3 ?,VDAC1 and NLRP3 in M1 macrophages were increased,while PP2Ac demethylation,p62 and Parkin were reduced(p<0.05).Compared with solvent control group,ABL 127 could further promote the expression of PP2Ac methylation,Beclin-1,LC3 ?/LC3 ?,VDAC1 and NLRP3 in M1 macrophages were further increased,while PP2Ac demethylation,p62 and Parkin were further decreased(p<0.05).Compared with the control siRNA Ml macrophage,after LCMT1 siRNA intervention,the expression of PP2Ac methylation,Beclin-l,LC3 ?/LC3 I,PINK1,VDAC1 and NLRP3 were decreased,while the expression of Parkin increased(p<0.05).3.The effect of PP2Ac methylation on M1 macrophage polarization via glycolysis metabolismMO macrophages differentiated into Ml macrophages,the activities of hexokinase,pyruvate kinase and lactate dehydrogenase were significantly increased(P<0.05).Compared with solvent control group,ABL127 could promote the activity of pyruvate kinase and lactate dehydrogenase(p<0.05),but have no effect on the activity of hexose kinase(p>0.05).Compared with the control siRNA M1 macrophage,the activity of HK,and LDH were decreased significantly after LCMT1 siRNA intervention(p<0.05).Conclusions:1)PP2Ac methylation promote the related genes expression of proinflamm-atory cytokines and chemokines such as COX-2,TNF-?,IL-6,CXCL-10 in M1 macrophages,and enhance the phagocytosis.2)PP2Ac methylation could protect against mitochondrial membrane potential and reduce intracellular ROS by enhance mitophagy in M1 macrophages.3)PP2Ac methylation could promote the M1 macrophages polarization via mitophagy promoting glycolysis metabolic enzyme activity.