DNA-PKcs Interacts With Aire And Regulates The Expression Of Toll-like Receptors In Macrophage

The autoimmune regulator (Aire) is a key transcriptional activator that mediatescentral tolerance in the thymus. Mutations in Aire can lead to APECED(autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy), a geneticautoimmune disease that manifests as autoimmunity to multiple endocrine glands. Inthe thymus, Aire is mainly expressed in medullary thymic epithelial cells (mTECs)and regulates the expression of peripheral tissue self-antigens (PTAs) in these cells.Although Aire is primarily expressed in the thymus, Aire gene transcripts have alsobeen detected in peripheral tissues, including the peripheral lymphoid organs, fetalliver, testis, and the ovaries. Moreover, Fuji et al have reported that Aire was alsoexpressed in CD14+dendritic cells (DCs), macrophages, granulocytes and B cells inthe peripheral blood.The role of Aire in the thymus is well illustrated, but its significance in theperiphery is still obscure. Recent studies have reported that the stromal cells of lymphnodes and the spleen that express Aire also contain a repertoire of PTAs transcripts,and they have the capacity to present transgene-encoded antigens directly to T cells,activating and ultimately deleting these T cells. We previously found that Aire, as atranscription factor, up-regulates expression of TLR1, TLR3and TLR8in Aire stablytransfected RAW264.7cells, may plays a crucial role in the recognition of pathogenicmicroorganisms and sustains peripheral immune tolerance in antigen presenting cells(APCs), but the mechanisms of these functions are unknown.There have been many indications that Aire is a transcriptional regulator, butseveral unusual features suggest that it does not operate as a classical transcriptionfactor by binding to promoters and driving mRNA initiation. Under normal conditions,Aire is found predominantly in punctate nuclear bodies. Aire can associate with a score of proteins and regulate the expression of target gene.DNA-dependent protein kinase (DNA-PK) is a Ser/Thr kinase that belongs to thephosphatidylinositol3-kinase (PI3K) family. DNA-PKcs is a catalytic subunit ofDNA-PK. DNA-PK can phosphorylate a number of proteins involved in transcription,and can modulate the transcription of many proteins. DNA-PK can interact with Aire,Maslovskaja J et al have initially identified in pull-down assays from Aire-transfectedmonocytes and confirmed in a broad mass spectrometry screen. DNA-PK canphosphorylate Aire in vitro and influences Aire's transactivation ability.In order to clarify the mechanisms of Aire to influence the expression of TLR1,TLR3and TLR8in RAW264.7cells, to discuss whether DNA-PK can interact withAire to regulate the expression of TLRs, our research on the basis of the model aboutthe Aire stably transfected RAW264.7cells and the Aire transiently transfectedprimary peritoneal macrophages, from the following several aspects of the research,the results as follow:1The interaction between DNA-PKcs and AireAt first, we used co-immunoprecipitation to determine whether Aire can interactwith endogenous DNA-PKcs in RAW264.7cells. The result showed that Aire canphysically associates with DNA-PKcs in RAW264.7cells.2The co-localization between Aire and DNA-PKcsIn order to detect the associates between Aire and DNA-PKcs, the intracellulardistribution of each protein was investigated by immunofluorescent microscopy. Theresult showed that intracellular co-localizations between Aire and part of DNA-PKcsin RAW264.7cells.3The effect of Aire on expression of TLRs after knockdown of DNA-PKcs inRAW264.7cellsIn order to research the effect of DNA-PKcs on TLRs expression which regulateby Aire in RAW264.7cells, after knockdown the DNA-PKcs in RAW264.7cells byDNA-PKcs siRNA, the expression of TLRs was examined by RT-qPCR and FCM.The results showed that knockdown of DNA-PKcs decreased the expression of TLR1, TLR3and TLR8in GFP-Aire/RAW cells, but not in pEGFPC1stably transfectedRAW264.7(GFP/RAW) cells. Expression of TLR2, TLR4, TLR5, TLR6, TLR7andTLR9did not change after DNA-PKcs siRNA treatment of GFP-Aire/RAW cells orcontrol GFP/RAW cells. The result indicated that DNA-PKcs can interact with Aire toregulate the expression of TLR1, TLR3and TLR8in RAW264.7cells.4The transcriptional activation of TLRs by Aire was decreased after knockdown ofDNA-PKcsIn order to further elucidate the effect of the interaction between DNA-PKcs andAire on TLRs's transcriptional activation, luciferase reporter assay was performed byTLRs reporter constructs. The result showed that the luciferase activity of the TLR1,TLR3, and TLR8promoters was significantly decreased after transfection withDNA-PKcs siRNA in GFP-Aire/RAW cells but was not changed in GFP/RAW cells,and there was no changed in the expression of TLR2, TLR5, TLR6, TLR7and TLR9in either cell line. The result indicated that DNA-PKcs can interact with Aire topromote the transcription of TLRs.5The effect of DNA-PKcs on expression of TLRs regulated by Aire in primaryperitoneal macrophagesIn order to illustrate the effect of DNA-PKcs in TLRs expression which regulateby Aire in primary peritoneal macrophages, after knockdown of DNA-PKcs byDNA-PKcs siRNA, we examined the expression of TLRs by RT-qPCR. The resultshowed after knockdown of DNA-PKcs, the mRNA levels of TLR1, TLR3and TLR8were decreased in pEGFPC1/Aire transiently transfected primary peritonealmacrophages, but were not changed in pEGFPC1transiently transfected macrophages,and no change in the expression of TLR2, TLR4, TLR5, TLR6, TLR7and TLR9wasdetected after knockdown in either cell line. The result further indicated thatDNA-PKcs can interact with Aire to regulate the expression of TLR1, TLR3andTLR8in macrophages.From the studies of above, the conclusions are:1Aire can interact with DNA-PKcs in RAW264.7cells. 2DNA-PKcs can interact with Aire to promote the expression and thetransactivation of TLR1, TLR3and TLR8in RAW264.7cells.3DNA-PKcs can also interact with Aire to promote the expression of TLR1,TLR3and TLR8in primary peritoneal macrophages.This study has demonstrate that DNA-PKcs can interact with Aire to regulate theexpression of TLR1, TLR3and TLR8in mouse macrophages and give theexperimental evidence for clarifying the mechanisms of Aire on the expression ofTLR1, TLR3and TLR8that may influence the recognition of pathogenicmicroorganisms or self-senescent and pathological cells. It could provide new clue forfurther the understanding of functions and significance of Aire in peripheralhematopoietic cells.