Role Of Gadd45a In CD4~+T Cell DNA Hypomethylation And In The Pathogenesis Of Systemic Lupus Erythematosus

Systemic lupus erythematosus(SLE) is a autoimmune disease which involved multiple organ systems.Its etiology and pathogenesis is unclear. Recent studies have shown that the T cell DNA demethylation play an important role in the pathogenesis of SLE.DNA methylation is one of many epigenetic factors that are important for cellular differentiation,gene regulation and genomic imprinting.In general,DNA hypomethylation is associated with gene transcription activation.Studies from Lu et al.showed that inhibiting T cell DNA methylation in vitro causes demethylation of regulatory sequences,contributes to increasing gene promoter activity,and thus leads to increasing gene expression.These genes include CD11a(ITGAL),perforin(PRF1),CD70(TNFSF7) and CD40 ligand(TNFSF5).Overexpression of these genes results in T cells autoreactivity,spontaneous monocyte/macrophage killing,B cell immunoglobulin overproduction.DNA methylation inhibitors could induce lupus-like autoimmunity in vitro and in vivo.Furthermore,T cells from patients with active lupus have genome-wide decrease in deoxymethylcytosin,and gene-specific hypomethylation.DNA hypomethylation induces autoimmune-related genes overexpression,and causes autoreactive monocyte/macrophage killing and excessive B cell stimulation.DNA demethylation also contributes to the striking female predilection of this disease.The mechanisms of demethylation in lupus T cells remain unknown.Recent study showed that Gadd45a can promote DNA repair,remove methylation marks,thereby reduce epigenetic silence of genes.Ultraviolet irradiation can upregulate expression of Gadd45a, and make SLE condition worse.Therefore,we propose the pathogenesis of SLE as follows:overexpression of nuclear protein Gadd45a can inhibit methylation at regulatory sequence of T cell methylation-sensitive genes, result in upregulated expression of the genes,then leads to spontaneous monocyte/macrophage killing and overstimulation of IgG synthesis, finally causes SLE.To prove the hypothesis,we used the following approach:firstly,to investigate the expression of Gadd45a in SLE CD4~+ T cells;secondly,to study the effect of UV on Gadd45a expression and DNA methylation;and thirdly,to examine the effect of upregulated and downregulated expression of Gadd45a on normal or SLE CD4~+ T cells. These studies would further reveal the mechanisms of demethylation of T cell genes in lupus,and provide a theoretical basis for more effective therapy of SLE. PartⅠExpression of Gadd45a in CD4~+ T cells from SLE patientsObjective:To investigate the expression of Gadd45a in SLE CD4~+T cells.Methods:Peripheral blood mononuclear cells(PBMCs) from 17 SLE patients and 10 healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation,CD4+ subsets were isolated by positive selection using Miltenyi beads.Expression of Gadd45a mRNA in CD4~+T cells was detected by real-time RT-PCR,and western blotting was used for protein expression in the same samples.The global methylation level was measured by DNA global methylation kit.The correlation between Gadd45a mRNA,global methylation level and systemic lupus erythematosus disease activity index(SLEDAI) were analyzed.Results:The expression of Gadd45a mRNA and protein in CD4+T cells from SLE patients were significantly elevated,Compared with controls(P＜0.01,P＜0.05,respectively).Global hypomethylation was observed in lupus CD4~+ T cells,compared with controls(P＜0.05).There was a significant negative correlation between Gadd45a mRNA expression and global methylation level(R=-0.676,P＜0.01).Furthermore, the Gadd45a mRNA level was positively correlated with increased disease activity in lupus patients as measured by SLEDAI(R=0.813,P ＜0.05).Conclusion:Gadd45a expression were significantly increased in lupus CD4~+ T cells,which is associated with demethylation and increased disease activity in SLE.PartⅡEffect of ultraviolet(UV) radiation on the expression of Gadd45a gene and methylation levels in Jurkat cells and CD4~+T cellsSectionⅠEffect of UV on the expression of Gadd45a gene and methylation levels in Jurkat cellsObjective:To explore the effect of UV on the expression of Gadd45a gene and methylation levels in Jurkat cells.Methods:Jurkat cells were irradiated with 1.0J/cm2 and 1.5J/cm2 UVB respectively,and collected at 6 hours,12 hours,24 hours and 48 hours after UVB irradiation.Real-time RT-PCR was used to detect the expression of Gadd45a mRNA and methylation sensitive genes CD11a, CD70 mRNA.Global methylation level was measured in the same samples. Results:(1) The expression of Gadd45a mRNA in Jurkat cells was significantly increased at 6 hours after UVB(1.0J/cm~2) irradiation (P＜0.01),accompanied with global demethylation((P＜0.05).The expression of CD11a,CD70 mRNA was upregulated,but the differences had no statistically significant.The expression of Gadd45a,CD11a,CD70 mRNA was signifcantly increased in Jurkat cells after 12 hours with irradiation UVB 1.0J/cm2(P＜0.05),and the global methylation level was significantly reduced(P＜0.05).24 hours and 48 hours after irradiation, Increased mRNA expression of Gadd45a,CD11a,CD70 mRNA and decreased global demethylation in Jurkat cells were also found at 24 and 48 hours after UVB(1.0J/cm~2) irradiation,but the differences had no statistically significant.(2) The expression of Gadd45a mRNA in Jurkat cells was significantly increased at 6,12,24 and 48 hours after UVB1.5 J/cm~2 irradiation,accompanied with significantly upregulated expression of CD11a,CD70 mRNA(P＜0.05 or P＜0.01).The global methylation level was significantly reduced at each time point after UVB1.5 J/cm~2 irradiation(P＜0.05 or P＜0.01).Conclusion:UVB can upregulate the expression of Gadd45a, CD11a and CD70 mRNA,and decrease global methylation in Jurkat cells SectionⅡEffect of UV on the expression of Gadd45a gene and methylation levels in CD4~+T cellsObjective:To explore the effect of UV on the expression of Gadd45a gene and methylation levels in CD4~+T cells.Methods:PBMCs from 3 healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation,CD4~+ subsets were isolated by positive selection using Miltenyi beads.CD4~+T cells were irradiated with 1.5J/cm2 UVB,and collected at 6 hours,24 hours,48 hours after UVB irradiation.Real-time RT-PCR was used to detect the expression of Gadd45a mRNA and methylation sensitive genes CD11a, CD70 mRNA,while Gadd45a protein expression and global methylation level were measured in the same samples.Results:The expression of Gadd45a mRNA was significantly increased in CD4~+T cells at 6 hours,24 hours,48 hours after irradiation UVB 1.5 J/cm2(P＜0.05),accompanied with significantly upregulated expression of CD11a,CD70 mRNA(P＜0.05).Expression of Gadd45a protein was also upregulated.Meanwhile,the global methylation level decreased at 48 hours after irradiation(P＜0.05).Conclusion:UVB can upregulate expression of Gadd45a,CD11a and CD70,and reduce global methylation in CD4~+T cells. PartⅢAbnormal Gadd45a gene expression and autoimmunitySectionⅠInducing T cells autoreactivity by overexpressing Gadd45aObjective:To investigate the effect of overexpression of Gadd45a by transfecting Gadd45a expressed plasmid on expression and methylation status of autoimmunity related genes and autoreactivity in normal CD4~+ T cells.Methods:Gadd45a expressed plasmid(PCDNA3.1-Gadd45a) and control plasmid(PCDNA3.1+) were constructed,then transfected into normal human CD4~+ T cells by transient electroporation.Real-time RT-PCR was used to detect the expression of Gadd45a,CD11a and CD70 mRNA.Cell proliferation assay and detection of IgG antibody production were performed following the protocol of kits.The global methylation level and methylation status of ITGAL promoter regulatory elements were detected by the methylation kit and sodium bisulfite sequencing, respectively.Results:Compared with CD4~+ T cells transfected control plasmid The expression of Gadd45a,CD11a and CD70 mRNA was upregulated in CD4~+ T cells transfected with PCDNA3.1-Gadd45a(P＜0.05).Increased cell proliferation activity and enhanced IgG production,and accompanied with global demethylation were also found in CD4~+T cells transfected with PCDNA3.1-Gadd45a(P＜0.05).The average methylation of CpG pairs in ITGAL promoter sequence(-1263 to-1111) was significantly reduced(P＜0.01),and six out of seven CpGs(-1263,-1255, -1191,-1159,-1121,-1111) were significantly demethylated in CD4~+ T cells transfected with PCDNA3.1-Gadd45a.Conclusion:Overexpression of Gadd45a can upregulate expression of methylation sensitive genes,and increase autoreactivity in CD4+T cells.SectionⅡInhibiting T cells autoreactivity by downregulating Gadd45a expressionObjective:To investigate the effect of downregulating Gadd45a expression by transfecting Gadd45a-siRNA on expression and methylation status of autoimmunity related genes,and autoreactivity in SLE CD4~+T cells.Methods:Gadd45a-siRNA was transfected into SLE CD4~+T cells by transient electroporation.Real-time RT-PCR was used to detect the expression of Gadd45a and methylation sensitive genes CD11a and CD70 mRNA.CDlla and CD70 protein levels were measured by flow cytometry.Cell proliferation assay and production of IgG antibody were performed following the protocol of kits.The global methylation level and methylation status of ITGAL promoter regulatory elements were detected by the methylation kit and sodium bisulfite sequencing, respectively.Results:The expression of Gadd45a,CD11a and CD70 mRNA were significantly downregulated in SLE CD4~+ T cells transfected with Gadd45a-siRNA(P＜0.05).Percent of CD11a staining cells and CD70 MFI(mean fluorescence intensity) were decreased too.Reduced cell proliferation activity and IgG production,and accompanied with global hypermethylation were also found in SLE CD4~+ T cells transfected with Gadd45a-siRNA.The average methylation of CpG pairs at ITGAL promoter sequence(-1263 to-1111) was significantly increased (P＜0.05),and four out of seven CpGs(-1263,-1223,-1159,-1121) were significantly hypermethylated in SLE CD4~+ T cells transfected with Gadd45a-siRNA.Conclusion:Downregulation of Gadd45a expression can inhibit expression of methylation sensitive genes,and reduce autoreactivity in SLE CD4~+T cells.