REG? Controls The Development Of Autoimmunity-Mechanistic Studies

REGy(also known as PSME3,PA28 Ki Antigen)is a member of the REG family of proteasomal activators,including REGa and REG?.According to previous studies REGy is located in the nucleus of the cell.Its functions are not limited to cell cycle regulation and apoptosis,also involved in the regulation of human disease and development,such as cancer,premature aging,fatty liver,obesity.Numerous studies have shown that,REGa and REG? can be induced by IFN-?,which regulae immune activity of proteasome and MHC-I antigen presentation process,while REG? has not been affectec by IFN-?.In addition,Ki antigen was found in the sera of patients with systemic lupus erythematosus and its relationship with autoimmune syndrome has not been elucidated.Therefore,REGy may be involved in the regulation of immuno-proteasome activity,antigen presentation process and regulation mechanism of autoimmune disease,remains a mystery.In this study,we examined the role of REGy knockout mice at different age and the effect on autoimmune disease by systematic observation and statistical analysis.We also demonstrate REG?'s regulatory mechanism in autoimmune disease via in-vitro and in-vivo experiments,using bioinformatics,pathology and other methodology.Results:As the REGy-deficient mice developed age associated autoimmune disease phenotypes in which antinuclear antibodies were increased in serum and renal damage was observed.Moreover,immune complex deposition occurred in glomerula.The infiltration cells of dendritic cells and macrophages were significantly increased in renal tissue with enlargement of the spleen and increased number of dendritic cells(DC)and CD8 + T cells.In addition,immune-proteasome levels of mRNA,protein,and activity,were up-regulated in primary kidney cells,bone marrow cell differentiation in DC(BMDCs)and murine spleen dendritic cells(DCs)from REGy deficient mice.Thus,REGy effect on antigen-presenting capacity of DC further regulates autoimmune diseases.The results of the above study suggest that REGymay affect the antigen presenting ability of DC by regulating the immuneproteasome,and the occurrence of autoimmune diseases.Finally,in order to identify the hypothesis,we used magnetic activated cell sorting of(MACS)CD8+T cells from OT-1 mouse with CFSE labeled to test cell proliferation in-vivo and in-vitro.The studies have shown that,using different concentration of OVA activated DC which was co-culture with CFSE labeled CD8+T cell,the CD8+T cell proliferation increased significantly higher with concentration dependent manner in REG? KO mice than REGy WT.Furthermore,using OVA(257-264)peptide,CD8+T cell proliferation was elevated with increasing concentration,but no difference was recorded in REG? WT and KO.The result demonstrate that REG?enhances antigen processing and improves the efficiency of antigen presentation,thus the REG? regulation involves in immuno-proteasome regulation.In the absence of REGy,immuno-proteasome LMP2 and LMP7 mRNA levels increase significantly by IFN-? induction,indicating that REG? negatively regulates immuno-proteasome.This further illustrates,REG? is not directly regulated the LMP2 and LMP7,but through the regulation of IFN? inducible protein to achieve.In a follow-up experiment,through degradation in-vitro and in-vivo experiments prove that REG? degradation by its new substrates—signal transduction and activator of transcription(P-STAT3)and implementation regulation of the LMP2 and LMP7.By gene silencing or inhibition of STAT3 phosphorylation or phosphorylation mimic-approaches,altered transcriptional activity of STAT3 can affect the expression of immuno-proteasome levels and activity.Furthermore,luciferase assay and chromatin immunoprecipitation experiments suggest that STAT3 is a transcription factor regulating the transcription of LMP2 and LMP7.In order to verify the above mechanism,we use methods of bioinformatics,statistics in human lupus nephritis REG? and immuno-proteasome mRNA expression levels.Additionally,histopathological analysis indicating that REGy-P-STAT3-LMP2/LMP7 signaling pathway may also be involved in human SLE,LN and RA.In conclusion,our findings provide the evidence that REGy regulates immuno-proteasome,and plays an important biological role in autoimmune regulation.Our finding of REG?-STAT3-immuno-proteasome-antigen signaling pathway provides a theoretical basis for the research of autoimmune diseases,particularly pential diagnostic marker of autoimmune diseases,therapeutic targets and drug development.