DNA-PKcs Interacts with Aire and Regulates the Expression of Toll-Like Receptors in RAW264.7 Cells
DNA-PKcs Interacts with Aire and Regulates the Expression of Toll-Like Receptors in RAW264.7 Cells
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DOI：
10.1111/j.1365-3083.2012.02682.x
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发表时间：
2012-05-01
影响因子：
3.7
通讯作者：
Li, Y.
中科院分区：
文献类型：
--
作者：
Wu, J.;Zhu, W.;Li, Y.
自身免疫调节因子(Aire)是中枢耐受外周组织自身抗原(PTAs)的关键介质，参与胸腺髓质上皮细胞(MTECs)中多种抗原的转录调控。然而，Aire在外周淋巴组织和造血细胞中的功能，特别是在单核细胞和巨噬细胞中的功能仍然知之甚少。我们先前发现，稳定表达Toll样受体(TLR1)1、TLR3和TLR8的RAW264.7(GFP-Aire/RAW)细胞中TLR1、TLR3和TLR8的表达显著上调，而其他TLRs的表达没有明显变化。Aire影响TLR1、TLR3和TLR8表达的机制尚不清楚。与其他蛋白质的相互作用，如DNA依赖的蛋白激酶(DNA-PK)，对调节Aire的转录活性至关重要。在本研究中，我们发现Aire和DNA-PK催化亚基(DNA-PKcs)共定位于GFP-Aire/RAW细胞的细胞核中，并且它们相互作用。在这些细胞中，DNA-PKcs的小干扰RNA下调TLR1、TLR3和TLR8的表达，但在稳定表达pEGFPC1的RAW264.7(GFP/RAW)细胞中没有观察到变化。DNA-PKcs基因敲除GFP-Aire/RAW或GFP/RAW细胞后，未观察到其他TLRs的表达变化。在瞬时转染pEGFPC1/Aire或pEGFPC1的原代腹膜巨噬细胞中也进行了类似的观察。利用荧光素酶活性测定，我们发现在GFP-Aire/RAW细胞中，DNA-PKcs被敲除后，TLR1、TLR3和TLR8启动子的转录活性也降低。综上所述，我们的结果提示DNA-PKcs可能与Aire相互作用，促进RAW264.7细胞中TLRs的表达。
The autoimmune regulator (Aire) is a key mediator of the central tolerance for peripheral tissue self-antigen (PTAs) and is involved in the transcriptional control of many antigens in thymic medullary epithelial cells (mTECs). However, the function of Aire in peripheral lymphoid tissues and haematopoietic cells, particularly in monocytes and macrophages, remains poorly understood. We previously found that the expression of Toll-like receptor (TLR) 1, TLR3 and TLR8 was notably upregulated in pEGFPC1/Aire stably transfected RAW264.7 (GFP-Aire/RAW) cells, while the expressions of other TLRs were not significantly changed. The mechanism by which Aire affects TLR1, TLR3 and TLR8 expression is not clear. Interactions with other proteins, such as DNA-dependent protein kinase (DNA-PK), are crucial for regulating the transcriptional activity of Aire. In this study, we found that Aire and DNA-PK catalytic subunit (DNA-PKcs) were co-located in the nucleus of GFP-Aire/RAW cells, and they interact with each other. Small interfering RNA knock-down of DNA-PKcs in these cells decreased the expression of TLR1, TLR3 and TLR8, but no change was observed in pEGFPC1 stably transfected RAW264.7 (GFP/RAW) cells. We did not observe any change in the expressions of other TLRs after DNA-PKcs knock-down in GFP-Aire/RAW or GFP/RAW cells. A similar observation has been made in pEGFPC1/Aire or pEGFPC1 transiently transfected primary peritoneal macrophages. Using a luciferase activity assay, we found the that the transcriptional activity of TLR1, TLR3 and TLR8 promoters was also decreased after knock-down of DNA-PKcs in GFP-Aire/RAW cells. In conclusion, our results suggest that DNA-PKcs may interact with Aire to promote the expression of TLRs in RAW264.7 cells.