Children's Foundation Research Institute at Le Bonheur Children's Hospital and Department of Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.
PLoS One. 2012;7(8):e43970. doi: 10.1371/journal.pone.0043970. Epub 2012 Aug 23.
As a part of the negative feedback mechanism, CpG DNA induces IRAK-M expression in monocytic cells. In the present study we investigated a biochemical signaling pathway and the transcription factors responsible for CpG DNA-mediated Irak-m gene expression. CpG DNA-induced Irak-m expression did not require new protein synthesis and was regulated at the transcriptional level through an endosomal pH-sensitive TLR9/MyD88 signaling pathway. Over-expression of the dominant negative (DN) form of or gene-specific knockdown of signaling modulators in the TLR9 pathway demonstrated that IRAK4, IRAK1, IRAK2, and PKD1 are required for Irak-m transcription induced by CpG DNA. Over-expression of DN-IRAK1 only partially, but significantly, inhibited CpG DNA-induced Irak-m promoter activity. While IRAK1 was critical for the initial phase, IRAK2 was required for the late phase of TLR9 signaling by sustaining activation of PKD1 that leads to activation of NF-κB and MAPKs. Irak-m promoter-luciferase reporters with alterations in the predicted cis-acting transcriptional regulatory elements revealed that the NF-κB consensus site in the Irak-m promoter region is absolutely required for Irak-m gene expression. AP-1 and CREB binding sites also contributed to the optimal Irak-m expression by CpG DNA. Collectively, our results demonstrate that IRAK2 plays a key role in the TLR9-mediated transcriptional regulation of Irak-m expression by sustaining activation of PKD1 and NF-κB.
作为负反馈机制的一部分，CpG DNA 在单核细胞中诱导 IRAK-M 的表达。在本研究中，我们研究了一条生化信号通路和负责 CpG DNA 介导 Irak-m 基因表达的转录因子。CpG DNA 诱导的 Irak-m 表达不需要新的蛋白质合成，并且通过内体 pH 敏感的 TLR9/MyD88 信号通路在转录水平上受到调节。TLR9 通路中信号调节剂的显性负（DN）形式的过度表达或基因特异性敲低表明，IRAK4、IRAK1、IRAK2 和 PKD1 是 CpG DNA 诱导的 Irak-m 转录所必需的。DN-IRAK1 的过度表达仅部分但显著抑制了 CpG DNA 诱导的 Irak-m 启动子活性。虽然 IRAK1 对初始阶段至关重要，但 IRAK2 通过维持 PKD1 的激活来维持 TLR9 信号的后期阶段，从而导致 NF-κB 和 MAPKs 的激活。对预测的顺式作用转录调节元件进行改变的 Irak-m 启动子-荧光素酶报告表明，Irak-m 启动子区域中的 NF-κB 共有结合位点对于 Irak-m 基因表达是绝对必需的。AP-1 和 CREB 结合位点也通过 CpG DNA 促进了 Irak-m 的最佳表达。总之，我们的结果表明，IRAK2 通过维持 PKD1 和 NF-κB 的激活，在 TLR9 介导的 Irak-m 表达的转录调节中发挥关键作用。
