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Interleukin-1 receptor-associated kinase 2- and protein kinase D1-dependent regulation of IRAK-monocyte expression by CpG DNA.

ABSTRACT: 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-?B and MAPKs. Irak-m promoter-luciferase reporters with alterations in the predicted cis-acting transcriptional regulatory elements revealed that the NF-?B 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-?B.

SUBMITTER: Kim YI

PROVIDER: S-EPMC3426515 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Interleukin-1 receptor-associated kinase 2- and protein kinase D1-dependent regulation of IRAK-monocyte expression by CpG DNA.

Kim Young-In YI Park Jeoung-Eun JE Kwon Ki Han KH Hong Cheol Yi CY Yi Ae-Kyung AK

PloS one 20120823 8

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 ...[more]

PMID: 22928050

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Project description:Specific microRNAs (miRNAs), small non-coding RNAs that support homeostatic gene expression, are significantly altered in abundance in human neurological disorders. In monocytes, increased expression of an NF-?B-regulated miRNA-146a down-regulates expression of the interleukin-1 receptor-associated kinase-1 (IRAK-1), an essential component of Toll-like/IL-1 receptor signaling. Here we extend those observations to the hippocampus and neocortex of Alzheimer disease (AD) brain and to stressed human astroglial (HAG) cells in primary culture. In 66 control and AD samples we note a significant up-regulation of miRNA-146a coupled to down-regulation of IRAK-1 and a compensatory up-regulation of IRAK-2. Using miRNA-146a-, IRAK-1-, or IRAK-2 promoter-luciferase reporter constructs, we observe decreases in IRAK-1 and increases in miRNA-146a and IRAK-2 expression in interleukin-1? (IL-1?) and amyloid-?-42 (A?42) peptide-stressed HAG cells. NF-?B-mediated transcriptional control of human IRAK-2 was localized to between -119 and +12 bp of the immediate IRAK-2 promoter. The NF-?B inhibitors curcumin, pyrrolidine dithiocarbamate or CAY10512 abrogated both IRAK-2 and miRNA-146a expression, whereas IRAK-1 was up-regulated. Incubation of a protected antisense miRNA-146a was found to inhibit miRNA-146a and restore IRAK-1, whereas IRAK-2 remained unaffected. These data suggest a significantly independent regulation of IRAK-1 and IRAK-2 in AD and in IL-1?+A?42 peptide-stressed HAG cells and that an inducible, NF-?B-sensitive, miRNA-146a-mediated down-regulation of IRAK-1 coupled to an NF-?B-induced up-regulation of IRAK-2 expression drives an extensively sustained inflammatory response. The interactive signaling of NF-?B and miRNA-146a further illustrate interplay between inducible transcription factors and pro-inflammatory miRNAs that regulate brain IRAK expression. The combinatorial use of NF-?B inhibitors with miRNA-146a or antisense miRNA-146a may have potential as a bi-pronged therapeutic strategy directed against IRAK-2-driven pathogenic signaling.

Project description:Kaposi sarcoma-associated herpesvirus (KSHV) is necessary but not sufficient for primary effusion lymphoma (PEL) development. Alterations in cellular signaling pathways are also a characteristic of PEL. Other B cell lymphomas have acquired an oncogenic mutation in the myeloid differentiation primary response 88 (MYD88) gene. The MYD88 L265P mutant results in the activation of interleukin-1 receptor associated kinase (IRAK). To probe IRAK/MYD88 signaling in PEL, we employed CRISPR/Cas9 technology to generate stable deletion clones in BCBL-1Cas9 and BC-1Cas9 cells. To look for off-target effects, we determined the complete exome of the BCBL-1Cas9 and BC-1Cas9 cells. Deletion of either MYD88, IRAK4, or IRAK1 abolished interleukin-1 beta (IL-1?) signaling; however, we were able to grow stable subclones from each population. Transcriptome sequencing (RNA-seq) analysis of IRAK4 knockout cell lines (IRAK4 KOs) showed that the IRAK pathway induced cellular signals constitutively, independent of IL-1? stimulation, which was abrogated by deletion of IRAK4. Transient complementation with IRAK1 increased NF-?B activity in MYD88 KO, IRAK1 KO, and IRAK4 KO cells even in the absence of IL-1?. IL-10, a hallmark of PEL, was dependent on the IRAK pathway, as IRAK4 KOs showed reduced IL-10 levels. We surmise that, unlike B cell receptor (BCR) signaling, MYD88/IRAK signaling is constitutively active in PEL, but that under cell culture conditions, PEL rapidly became independent of this pathway.IMPORTANCE One hundred percent of primary effusion lymphoma (PEL) cases are associated with Kaposi sarcoma-associated herpesvirus (KSHV). PEL cell lines, such as BCBL-1, are the workhorse for understanding this human oncovirus and the host pathways that KSHV dysregulates. Understanding their function is important for developing new therapies as well as identifying high-risk patient groups. The myeloid differentiation primary response 88 (MYD88)/interleukin-1 receptor associated kinase (IRAK) pathway, which has progrowth functions in other B cell lymphomas, has not been fully explored in PEL. By performing CRISPR/Cas9 knockout (KO) studies targeting the IRAK pathway in PEL, we were able to determine that established PEL cell lines can circumvent the loss of IRAK1, IRAK4, and MYD88; however, the deletion clones are deficient in interleukin-10 (IL-10) production. Since IL-10 suppresses T cell function, this suggests that the IRAK pathway may serve a function in vivo and during early-stage development of PEL.

Dataset Information

Interleukin-1 receptor-associated kinase 2- and protein kinase D1-dependent regulation of IRAK-monocyte expression by CpG DNA.

Publications

Interleukin-1 receptor-associated kinase 2- and protein kinase D1-dependent regulation of IRAK-monocyte expression by CpG DNA.

Similar Datasets

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