| SMYD3(protein 3 containing SET and MYND structural domains)belongs to the SMYD methylesterase family and is a histone lysine methyltransferase that promotes gene transcription mainly by catalysing the trimethylation of lysine at position 4 of histone subunit 3(H3K4me3).Studies have shown that SMYD3 plays a key role in tumour cell proliferation and differentiation;however,its role in macrophage polarization is unclear.Objective:This study explores the mechanism through which the histone methyltransferase SMYD3 affects the polarization of macrophages by regulating their internal metabolism.Methods:1.GSE95405 data were downloaded from the GEO database(GSE95405 was assayed using the Affymetrix Human Genome U133 Plus2.0 platform containing cytokine-induced transcriptome data for classically polarized M1 and M2 macrophages)and bioinformatics analysis was performed to obtain differences between M1 and M2 macrophage expressed genes.Then,M1 and M2 macrophage models were established in vitro by cytokine induction method,and the expression of M1 macrophage markers and M2 macrophage markers were detected using qRT-PCR method,and the screened differential histone modifier genes were validated using qRT-PCR method.2.SMYD3 overexpression vector was constructed in M1 macrophages to investigate whether its methyltransferase activity was involved in LPS/IFN-γ-induced macrophage activation.Changes in SMYD3 and H3K4me3 protein levels were analyzed by western blotting,and changes in fluorescence intensity of H3K4me3 were detected by immunofluorescence.The mRNA levels of CCR7,CD80,HLADR,TNF-α and CXCL16 were detected by qRT-PCR in M1 macrophages transfected with SMYD3 overexpression vector and null control.Flow cytometry analysis of protein expression levels of CCR7,CD80 and HLADR in M1 macrophages transfected with SMYD3 overexpression vector and null control.3.RNA-seq analysis was performed on M1 macrophages transfected with SMYD3 overexpression vector,and GO enrichment analysis and KEGG enrichment analysis were performed for differentially expressed genes.mRNA expression levels of some key metabolic genes in M1 macrophages in control and overexpression SMYD3 groups were verified by qRT-PCR.4.M1 macrophages were transfected with SMYD3 overexpression vector and null,and ChIP was performed on each sample using anti-H3K4me3 antibody,followed by genome-wide ChIP-seq analysis of the antibody co-precipitated DNA.Next,GO enrichment analysis was performed on Peak neighboring genes.IGV(Integrative Genomics Viewer)profiles of the H3K4me3-enriched regions on the SHMT2,MTHFD2L,MTHFD1L,ALDH1L2,MTR,ATIC and MTHFD1 motifs were analyzed and verified by ChIP-qPCR in the promoter region of the MTHFD1L gene H3K4me3 enrichment differences in the promoter region of MTHFD1L gene were verified by ChIP-qPCR.5.Two siRNAs(si-MTHFD1L#1 and si-MTHFD1L#2)were used to knock down MTHFD1L expression in M1 macrophages,and then the expression changes of M1-related genes were detected by qRT-PCR after knocking down MTHFD1L in M1 macrophages.In addition,after knocking down MTHFD1L,M1 macrophages were stimulated with exogenous addition of low concentration of formate,and the expression changes of M1-related genes were analyzed by qRT-PCR and flow cytometry.6.ELISA was performed to detect the changes in supernatant and intracellular formic acid content in M1 macrophages in control and MTHFD1L knockdown groups.In addition,changes in supernatant and intracellular formate content of M1 macrophages in the control and SMYD3 high expression groups were detected by ELISA.M1 macrophages were stimulated exogenously with formate(0,2,10 and 40mM)and changes in mRNA expression levels of CCR7,CD80,HLADR,TNF-αand CXCL16 were detected by qRT-PCR.Protein expression changes of CCR7 and HLADR were analyzed by flow cytometry.7.RNA-seq data were further analyzed for the expression of key genes of the mitochondrial respiratory chain complex,including MT-ND1,MT-ND2,MT-ND3(respiratory chain complex Ⅰ),SDHA(respiratory chain complex Ⅱ),MT-CYTB,UQCRC1(respiratory chain complex Ⅲ),MT-CO1(respiratory chain complex Ⅳ)and ATP6(respiratory chain complex Ⅴ)genes.M1 macrophages were stimulated with different concentrations of formic acid.qRT-PCR was performed to detect changes in gene expression of ND1,SDHA,CYTB,COX1 and ATP6.The H3K4me3 modification status of the promoters of MT-ND1,MT-ND2,MT-ND3,SDHA,MTCYTB,UQCRC1,MT-CO1 and ATP6 genes were further examined by ChIP-seq analysis.8.Formate(0,2,10 and 40mM)stimulated M1 macrophages and detected ROS production by flow cytometry.Western blot detected LC3,PINK 1 and pParkin/Parkin protein expression levels.Immunofluorescence was used to detect changes in LC3,PINK1 and p-Parkin fluorescence intensity.Results:1.The results showed that GSE95405 obtained a total of 2139 differentially expressed genes,of which 903 were upregulated in M1-type macrophages and 1236 were upregulated in M2-type macrophages.The differentially expressed genes obtained between M1 and M2 type macrophages were intersected with the set of histone modifying enzyme genes we obtained and a total of 10 differentially expressed histone modifying enzyme genes were obtained,including 3 up-regulated genes and 7 down-regulated genes in M1.The expression of M1 macrophage markers and M2 macrophage markers were detected using qRT-PCR method.The results showed that M1-type macrophages induced with 100ng/mL LPS,20ng/mL IFN-γ and M2-type macrophages induced with 25ng/mL IL-4 and 25ng/mL IL-13 all grew in clusters against the wall.The former induced high expression of CCR7,CD80,HLADR,TNF-α,CXCL16 in macrophages,and the latter induced high expression of CD206,PPARG,CDllb,VEGFA,CCL22 in macrophages.The mRNA expression levels were significantly higher in M2 macrophages stimulated with IL-4 and IL-13.2.Overexpression of SMYD3 in M1-type macrophages significantly increased the expression level of H3K4me3.In addition,overexpression of SMYD3 in M1 macrophages significantly suppressed the mRNA expression levels of CCR7,CD80,HLADR,TNF-α,and CXCL16 compared to the null control.Flow cytometry analysis of the changes in the protein expression levels of CCR7,CD80 and HLADR showed that the protein expression levels of CCR7,CD80 and HLADR were also significantly reduced in M1 macrophages after overexpression of SMYD3.3.RNA-seq results showed that a total of 1094 genes were down-regulated and 843 genes were up-regulated in the M1 macrophage overexpression SMYD3 group compared with the null control.The results of GO enrichment analysis showed that many metabolic pathways were enriched in the SMYD3 overexpression group of M1 macrophages,and the results of KEGG enrichment analysis further showed that folate metabolic pathways were highly enriched and many key metabolic pathways in the SMYD3 overexpression group of M1 macrophages were highly enriched.KEGG enrichment analysis further showed that folate metabolic pathway was highly enriched and many key metabolic genes SHMT2,MTHFD2L,MTHFD1L,ALDH1L2 were significantly upregulated in SMYD3 overexpression group.qRTPCR results showed that,except for ATIC,SHMT2,MTHFD2L,MTHFD1L,ALDH1L2,MTR and MTHFD1 mRNA levels were significantly upregulated in SMYD3 overexpression group in line with RNA-seq results.MTHFD1 mRNA levels were significantly up-regulated in the overexpressed SMYD3 group.4.The signal distribution map near the genes showed strong signal peaks appeared near TSS,indicating that Reads were mainly enriched near TSS.peak center signal heat map showed a more concentrated signal near the enriched sites.The functional region Peak distribution map shows that most of the regulatory factors bind in the promoter region.The results of GO enrichment analysis show that most of the enriched pathways are related to metabolic processes.the IGV of H3K4me3 enriched regions on SHMT2,MTHFD2L,MTHFDIL,ALDH1L2,MTR,ATIC and MTHFD1 motifs(Integrative Genomics Viewer)mapping results showed that the H3K4me3 binding peak around the TSS region of MTHFD1L was significantly elevated in the SMYD3 overexpression group compared to the null control,while the other genes were not significantly different between the two groups.ChIP-qPCR results showed that the SMYD3 overexpression group had significantly higher MTHFD1L expression was significantly elevated in the SMYD3 overexpression group.5.mRNA expression levels of CCR7,CD80,HLADR,TNF-α and CXCL16 were significantly increased in the MTHFD1L knockdown group compared with the control group.Exogenous addition of low concentrations of formate to stimulate M1 macrophages after knockdown of MTHFD1L showed that the M1 phenotype promoted by MTHFD1L knockdown was reversed by exogenous addition of formate.Flow cytometry analysis of CCR7,CD80,HLADR protein expression also showed consistent changes.6.Knockdown of MTHFD1L significantly reduced both supernatant and intracellular formic acid content in M1 macrophages compared to controls.Overexpression of SMYD3 significantly increased cell supernatant and intracellular formic acid content.Macrophages were stimulated with different concentrations(2,10 and 40mM)of formic acid,and the results showed that exogenous formic acid stimulation significantly inhibited the expression of M1-related genes.Flow cytometry analysis of protein expression changes of CCR7,CD80,HLADR after stimulation with different concentrations of formic acid showed that the protein expression levels of CCR7,CD80,HLADR were differentially inhibited.7.RNA-seq results showed that the mitochondrial respiratory chain complex genes,except SDHA,showed a consistent elevation trend in the SMYD3 overexpression group.Stimulation of M1 macrophages with different concentrations of formic acid showed that formic acid stimulation significantly increased the expression of ND1(respiratory chain complex Ⅰ),CYTB(respiratory chain complexⅢ),COX1(respiratory chain complex Ⅳ)and ATP6(respiratory chain complex Ⅴ),but not the expression of SDHA,the gene of complex Ⅱ.IGV mapping of the H3K4me3-enriched region on the locus showed that SDHA,UQCRC1 was transcriptionally regulated by H3K4me3,but overexpression of SMYD3 did not increase the transcriptional activity of its promoter region,and other genes were shown not to be transcriptionally regulated by H3K4me3.8.Western blotting analysis showed that formic acid(2,10 and 40mM)stimulation significantly increased the signal intensity of LC3,PINK1 and p-Parkin.Immunofluorescence results showed that formic acid treatment significantly increased LC3,PINK1 and p-Parkin fluorescence intensities.Conclusions:Our results suggest that histone methyltransferase SMYD3 causes accumulation of the intracellular metabolite formic acid by activating the activity of the metabolic enzyme MTHFD1L in the one-carbon folate metabolic pathway,and that accumulation of formic acid increases ROS production and promotes mitochondrial respiratory chain complex activity thereby inducing mitochondrial autophagy and inhibiting M1 macrophage polarization. |
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