Minjia Yu

IRAK‐M mediates Toll‐like receptor/IL‐1R‐induced NFκB activation and cytokine production

Toll‐like receptors transduce their signals through the adaptor molecule MyD88 and members of the IL‐1R‐associated kinase family (IRAK‐1, 2, M and 4). IRAK‐1 and IRAK‐2, known to form Myddosomes with MyD88–IRAK‐4, mediate TLR7‐induced TAK1‐dependent NFκB activation. IRAK‐M was previously known to function as a negative regulator that prevents the dissociation of IRAKs from MyD88, thereby inhibiting downstream signalling. However, we now found that IRAK‐M was also able to interact with MyD88–IRAK‐4 to form IRAK‐M Myddosome to mediate TLR7‐induced MEKK3‐dependent second wave NFκB activation, which is uncoupled from post‐transcriptional regulation. As a result, the IRAK‐M‐dependent pathway only induced expression of genes that are not regulated at the post‐transcriptional levels (including inhibitory molecules SOCS1, SHIP1, A20 and IκBα), exerting an overall inhibitory effect on inflammatory response. On the other hand, through interaction with IRAK‐2, IRAK‐M inhibited TLR7‐mediated production of cytokines and chemokines at translational levels. Taken together, IRAK‐M mediates TLR7‐induced MEKK3‐dependent second wave NFκB activation to produce inhibitory molecules as a negative feedback for the pathway, while exerting inhibitory effect on translational control of cytokines and chemokines.

Inactivation of the Enzyme GSK3α by the Kinase IKKi Promotes AKT-mTOR Signaling Pathway that Mediates Interleukin-1-Induced Th17 Cell Maintenance

Interleukin-1 (IL-1)-induced activation of the mTOR kinase pathway has major influences on Th17 cell survival, proliferation, and effector function. Via biochemical and genetic approaches, the kinases IKKi and GSK3α were identified as the critical intermediate signaling components for IL-1-induced AKT activation, which in turn activated mTOR. Although insulin-induced AKT activation is known to phosphorylate and inactivate GSK3α and GSK3β, we found that GSK3α but not GSK3β formed a constitutive complex to phosphorylate and suppress AKT activation, showing that a reverse action from GSK to AKT can take place. Upon IL-1 stimulation, IKKi was activated to mediate GSK3α phosphorylation at S21, thereby inactivating GSK3α to promote IL-1-induced AKT-mTOR activation. Thus, IKKi has a critical role in Th17 cell maintenance and/or proliferation through the GSK-AKT-mTOR pathway, implicating the potential of IKKi as a therapeutic target.

MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases.

MyD88-dependent GM-CSF production by endothelial cells plays a role in the initiation of obesity-associated inflammation by promoting adipose macrophage recruitment and M1-like polarization.

Pellino 2 is critical for Toll-like receptor/Interleukin-1 receptor (TLR/IL-1R)-mediated post-transcriptional control

Background: Pellino proteins are involved in polyubiquitination and degradation of IRAK1, a key molecule in TLR/IL-1R-mediated signaling. Results: Pellino 2 knockdown reduced TAK1-dependent NFκB activation and inflammatory gene mRNA stabilization. Conclusion: Pellino 2 plays a critical role for TLR/IL-1R-mediated post-transcriptional control. Significance: Pellino 2 is the critical signaling molecule controlling TAK1- and MEKK3-dependent pathways and also plays a critical role for TLR/IL-1R-mediated post-transcriptional control. Interleukin 1 receptor-associated kinase 1(IRAK1), a key molecule in TLR/IL-1R-mediated signaling, is phosphorylated, ubiquitinated, and degraded upon ligand stimulation. We and others have recently identified Pellino proteins as novel RING E3 ubiquitin ligases involved in IRAK1 polyubiquitination and degradation. However, it remains unclear how each Pellino member distinctly regulates TLR/IL-1R signaling by modulating IRAK1 ubiquitination. In this study we examined the role of Pellino 2 in IL-1- and LPS-mediated signaling and gene expression by knocking down Pellino 2 in human 293-IL-1R cells and primary bone marrow macrophages. Pellino 2 (but not Pellino 1) knockdown abolished IL-1- and LPS-induced Lys-63-linked IRAK1 ubiquitination with reduced Lys-48-linked IRAK1 ubiquitination. Furthermore, Pellino 2 is required for TAK1-dependent NFκB activation. However, because of the retained TAK1-independent NFκB activation, the levels of IL-1- and LPS-induced NFκB activation were not substantially affected in Pellino 2 knockdown 293-IL-1R cells and primary macrophages, respectively. On the other hand, Pellino 2 knockdown reduced the IL-1- and LPS-induced inflammatory gene expression at late time points, which was accompanied by increased decay rates of the mRNAs of the inflammatory genes. Importantly, IL-1- and LPS-mediated JNK and ERK activation were substantially attenuated in Pellino 2 knock-down cells, implicating MAPK activation in TLR/IL-1R-induced mRNA stabilization. Taken together, this study demonstrated that Pellino 2 plays a critical role for TLR/IL-1R-mediated post-transcriptional control.

β-TrCP-Mediated IRAK1 Degradation Releases TAK1-TRAF6 from the Membrane to the Cytosol for TAK1-Dependent NF-κB Activation

ABSTRACT Interleukin-1 (IL-1) receptor-associated kinase (IRAK1) is phosphorylated, ubiquitinated, and degraded upon IL-1 stimulation. IRAK1 can be ubiquitinated through both K48- and K63-linked polyubiquitin chains upon IL-1 stimulation. While the Pellino proteins have been shown to meditate K63-linked polyubiquitination on IRAK1, the E3 ligase for K48-linked ubiquitination of IRAK1 has not been identified. In this study, we report that the SCF (Skp1–Cullin1–F-box)–β-TrCP complex functions as the K48-linked ubiquitination E3 ligase for IRAK1. IL-1 stimulation induced the interaction of IRAK1 with Cullin1 and β-TrCP. Knockdown of β-TrCP1 and β-TrCP2 attenuated the K48-linked ubiquitination and degradation of IRAK1. Importantly, β-TrCP deficiency abolished the translocation TAK1-TRAF6 complex from the membrane to the cytosol, resulting in a diminishment of the IL-1-induced TAK1-dependent pathway. Taken together, these results implicate a positive role of β-TrCP-mediated IRAK1 degradation in IL-1-induced TAK1 activation.

Interleukin-1 Receptor-Associated Kinase-2 (IRAK2) Is a Critical Mediator of Endoplasmic Reticulum (ER) Stress Signaling

Endoplasmic reticulum (ER) stress occurs when unfolded proteins accumulate in the lumen of the organelle, triggering signal transduction events that contribute either to cellular adaptation and recovery or alternatively to cellular dysfunction and death. ER stress has been implicated in numerous diseases. To identify novel modulators of ER stress, we undertook a siRNA library screen of the kinome, revealing Interleukin-1 Receptor-Associated Kinase-2 (IRAK2) as a contributor to unfolded protein response (UPR) signaling and ER stress-induced cell death. Knocking down expression of IRAK2 (but not IRAK1) in cultured mammalian cells suppresses ER stress-induced expression of the pro-apoptotic transcription factor CHOP and activation of stress kinases. Similarly, RNAi-mediated silencing of the IRAK family member Tube (but not Pelle) suppresses activation of stress kinase signaling induced by ER stress in Drosophila cells. The action of IRAK2 maps to the IRE1 pathway, rather than the PERK or ATF6 components of the UPR. Interestingly, ER stress also induces IRAK2 gene expression in an IRE1/XBP1-dependent manner, suggesting a mutually supporting amplification loop involving IRAK2 and IRE1. In vivo, ER stress induces Irak2 expression in mice. Moreover, Irak2 gene knockout mice display defects in ER stress-induced CHOP expression and IRE1 pathway signaling. These findings demonstrate an unexpected linkage of the innate immunity machinery to UPR signaling, revealing IRAK2 as a novel amplifier of the IRE1 pathway.

IRAKM-Mincle axis links cell death to inflammation: Pathophysiological implications for chronic alcoholic liver disease

Lipopolysaccharide (LPS)‐mediated activation of Toll‐like receptors (TLRs) in hepatic macrophages and injury to hepatocytes are major contributors to the pathogenesis of alcoholic liver disease. However, the mechanisms by which TLR‐dependent inflammatory responses and alcohol‐induced hepatocellular damage coordinately lead to alcoholic liver disease are not completely understood. In this study, we found that mice deficient in interleukin‐1 receptor‐associated kinase M (IRAKM), a proximal TLR pathway molecule typically associated with inhibition of TLR signaling, were actually protected from chronic ethanol‐induced liver injury. In bone marrow‐derived macrophages challenged with low concentrations of LPS, which reflect the relevant pathophysiological levels of LPS in both alcoholic patients and ethanol‐fed mice, the IRAKM Myddosome was preferentially formed. Further, the IRAKM Myddosome mediated the up‐regulation of Mincle, a sensor for cell death. Mincle‐deficient mice were also protected from ethanol‐induced liver injury. The endogenous Mincle ligand spliceosome‐associated protein 130 (SAP130) is a danger signal released by damaged cells; culture of hepatocytes with ethanol increased the release of SAP130. Ex vivo studies in bone marrow‐derived macrophages suggested that SAP130 and LPS synergistically activated inflammatory responses, including inflammasome activation. Conclusion: This study reveals a novel IRAKM‐Mincle axis that contributes to the pathogenesis of ethanol‐induced liver injury. (Hepatology 2016;64:1978‐1993).

The Dual Functions of IL-1 Receptor-Associated Kinase 2 in TLR9-Mediated IFN and Proinflammatory Cytokine Production

Bone marrow-derived plasmacytoid dendritic cells (pDCs) from IL-1R–associated kinase (IRAK)2-deficient mice produced more IFNs than did wild-type pDCs upon stimulation with the TLR9 ligand CpG. Furthermore, in CpG-stimulated IRAK2-deficient pDCs there was increased nuclear translocation of IFN regulatory factor 7, the key transcription factor for IFN gene transcription in these cells. In IRAK2-deficient macrophages, enhanced NF-κB activation and increased expression of CpG-induced genes were detected within 2 h after treatment. However, at later times, NF-κB activation was decreased and, in contrast to the results with IFN, there was less secretion of other proinflammatory cytokines (such as TNF-α) and chemokines in CpG-stimulated IRAK2-deficient pDCs and macrophages. Therefore, although IRAK2 is a negative regulator of TLR9-mediated IFN production through its modulation of the transcriptional activity of IFN regulatory factor 7, it is also a positive regulator of TLR9-mediated proinflammatory cytokine and chemokine production at some level subsequent to transcription.

Abstract 2928: Interleukin-1 receptor-associated kinase-2 (IRAK2) is a critical mediator of endoplasmic reticulum (ER) stress.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Endoplasmic reticulum (ER) stress occurs when unfolded proteins accumulate in the lumen of the organelle, triggering signal transduction events that contribute either to cellular adaptation and recovery or alternatively to cellular dysfunction and death. ER stress has been implicated in numerous diseases, including cancer where harsh tumor microenvironments induce ER stress and create pressure for evolution of malignant clones that have acquired mechanisms for successful adaptation to stressful environments. To identify novel modulators of ER stress, we undertook a siRNA library screen of the kinome, revealing Interleukin-1 Receptor-Associated Kinase-2 (IRAK2) as a contributor to unfolded protein response (UPR) signaling and ER stress-induced cell death. Knocking down expression of IRAK2 (but not IRAK1) in cultured mammalian cells suppresses ER stress-induced expression of the pro-apoptotic transcription factor CHOP and activation of stress kinases. Similarly, RNAi-mediated silencing of the IRAK family member Tube (but not Pelle) suppresses activation of stress kinase signaling induced by ER stress in Drosophila cells. The action of IRAK2 maps to the IRE1 pathway, rather than the PERK or ATF6 components of the UPR. Interestingly, ER stress also induces IRAK2 gene expression in an IRE1/XBP1-dependent manner, suggesting a mutually supporting amplification loop involving IRAK2 and IRE1. In vivo, ER stress induces Irak2 expression in mice. Moreover, Irak2 gene knockout mice display defects in ER stress-induced CHOP expression and IRE1 pathway signaling. These findings demonstrate an unexpected linkage of the innate immunity machinery to UPR signaling, revealing IRAK2 as a novel amplifier of the IRE1 pathway involved in ER stress signaling. Future analysis of the role of IRAK2 in animal models of cancer will aid in assessing whether IRAK2 provides a novel targets for potential cancer therapeutics development. (Supported by NIH grant AG-15393). Citation Format: Ravanan Palaniyandi, Samir Benosman, Ying-Chen Hou, Ricardo G. Correa, Minjia Yu, Muhammet Fatih Gulen, Xiaoxia Li, James Thomas, Yasuko Matsuzawa, Shu-Ichi Matsuzawa, John C. Reed. Interleukin-1 receptor-associated kinase-2 (IRAK2) is a critical mediator of endoplasmic reticulum (ER) stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2928. doi:10.1158/1538-7445.AM2013-2928 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.