Tristetraprolin regulates necroptosis during tonic Toll-like receptor 4 (TLR4) signaling in murine macrophages

The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88−/−, Trif−/−, MyD88−/−Trif−/−, MK2−/−, and Zfp36−/− mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)–induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.

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Endotoxin Tolerance Inhibits Lyn and c-Src Phosphorylation and Association with Toll-Like Receptor 4 but Increases Expression and Activity of Protein Phosphatases

Journal of Innate Immunity ◽

10.1159/000440838 ◽

2015 ◽

Vol 8 (2) ◽

pp. 171-184 ◽

Cited By ~ 6

Author(s):

Yanbao Xiong ◽

Michael Murphy ◽

Tissa T. Manavalan ◽

Goutham Pattabiraman ◽

Fu Qiu ◽

...

Keyword(s):

Protein Tyrosine Phosphatase ◽

Protein Phosphatase ◽

Tyrosine Phosphatase ◽

Endotoxin Tolerance ◽

Mitogen Activated Protein Kinase ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling ◽

Mitogen Activated Protein ◽

Ptp 1B

Endotoxin tolerance protects the host by limiting excessive 'cytokine storm' during sepsis, but compromises the ability to counteract infections in septic shock survivors. It reprograms Toll-like receptor (TLR) 4 responses by attenuating the expression of proinflammatory cytokines without suppressing anti-inflammatory and antimicrobial mediators, but the mechanisms of reprogramming remain unclear. In this study, we demonstrate that the induction of endotoxin tolerance in human monocytes, THP-1 and MonoMac-6 cells inhibited lipopolysaccharide (LPS)-mediated phosphorylation of Lyn, c-Src and their recruitment to TLR4, but increased total protein phosphatase (PP) activity and the expression of protein tyrosine phosphatase (PTP) 1B, PP2A, PTP nonreceptor type (PTPN) 22 and mitogen-activated protein kinase phosphatase (MKP)-1. Chemical PP inhibitors, okadaic acid, dephostatin and cantharidic acid markedly decreased or completely abolished LPS tolerance, indicating the importance of phosphatases in endotoxin tolerization. Overexpression of PTPN22 decreased LPS-mediated nuclear factor (NF)-κB activation, p38 phosphorylation and CXCL8 gene expression, while PTPN22 ablation upregulated LPS-induced p65 NF-κB and p38 phosphorylation and the expression of TNF-a and pro-IL-1ß mRNA, indicating PTPN22 as an inhibitor of TLR4 signaling. Thus, LPS tolerance interferes with TLR4 signaling by inhibiting Lyn and c-Src phosphorylation and their recruitment to TLR4, while increasing the phosphatase activity and expression of PP2A, PTPN22, PTP1B and MKP1.

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The Small GTPase Arf6 Is Essential for the Tram/Trif Pathway in TLR4 Signaling

Journal of Biological Chemistry ◽

10.1074/jbc.m113.499194 ◽

2013 ◽

Vol 289 (3) ◽

pp. 1364-1376 ◽

Cited By ~ 19

Author(s):

Tim Van Acker ◽

Sven Eyckerman ◽

Lieselotte Vande Walle ◽

Sarah Gerlo ◽

Marc Goethals ◽

...

Keyword(s):

Plasma Membrane ◽

Critical Role ◽

Adaptor Proteins ◽

Small Gtpase ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling ◽

Mouse Macrophages ◽

Adp Ribosylation Factor ◽

Irf3 Activation

Recognition of lipopolysaccharides (LPS) by Toll-like receptor 4 (TLR4) at the plasma membrane triggers NF-κB activation through recruitment of the adaptor proteins Mal and MyD88. Endocytosis of the activated TLR4 allows recruitment of the adaptors Tram and Trif, leading to activation of the transcription factor IRF3 and interferon production. The small GTPase ADP-ribosylation factor 6 (Arf6) was shown to regulate the plasma membrane association of Mal. Here we demonstrate that inhibition of Arf6 also markedly reduced LPS-induced cytokine production in Mal−/− mouse macrophages. In this article, we focus on a novel role for Arf6 in the MyD88-independent TLR4 pathway. MyD88-independent IRF3 activation and IRF3-dependent gene transcription were strictly dependent on Arf6. Arf6 was involved in transport of Tram to the endocytic recycling compartment and internalization of LPS, possibly explaining its requirement for LPS-induced IRF3 activation. Together, these results show a critical role for Arf6 in regulating Tram/Trif-dependent TLR4 signaling.

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Common Interaction Surfaces of the Toll-Like Receptor 4 Cytoplasmic Domain Stimulate Multiple Nuclear Targets

Molecular and Cellular Biology ◽

10.1128/mcb.23.7.2543-2555.2003 ◽

2003 ◽

Vol 23 (7) ◽

pp. 2543-2555 ◽

Cited By ~ 45

Author(s):

Tapani Ronni ◽

Vishal Agarwal ◽

Michael Haykinson ◽

Margaret E. Haberland ◽

Genhong Cheng ◽

...

Keyword(s):

Target Genes ◽

Interleukin 1 ◽

Adaptor Proteins ◽

Critical Surface ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling ◽

Tir Domain ◽

Anti Inflammatory ◽

Mutant Phenotypes

ABSTRACT Toll-like receptor 4 (TLR4) mediates the host response to lipopolysaccharide (LPS) by promoting the activation of pro- and anti-inflammatory cytokine genes. To activate each gene, numerous signal transduction pathways are required. The adaptor proteins MyD88 and TIRAP contribute to the activation of several and possibly all pathways via direct interactions with TLR4's Toll/interleukin-1 receptor (IL-1R) (TIR) domain. However, additional adaptors that are required for the activation of specific subsets of pathways may exist, which could contribute to the differential regulation of target genes. Furthermore, it remains unknown whether direct interactions that have been reported between TIR domains and other proteins are required for TLR4 signaling. To address these issues, we systematically mutated the TLR4 TIR domain in the context of a CD4/TLR4 fusion protein. Several exposed residues defining at least two structural surfaces were required in macrophages for activation of the proinflammatory IL-12 p40 and anti-inflammatory IL-10 promoters, as well as promoters dependent on individual transcription factors. Interestingly, the same residues were required by all promoters tested, suggesting that the signaling pathways diverge downstream of the adaptors. The mutant phenotypes provide a framework for future studies of TLR4 signaling, as the interaction supported by each critical surface residue will need to be defined.

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Absence of Toll-Like Receptor 4 Signaling Results in Delayed Yersinia enterocolitica YopP-Induced Cell Death of Dendritic Cells

Infection and Immunity ◽

10.1128/iai.00756-06 ◽

2006 ◽

Vol 75 (1) ◽

pp. 512-517 ◽

Cited By ~ 11

Author(s):

Sabine Gröbner ◽

Sebastian Schulz ◽

Irena Soldanova ◽

Dani S. J. Gunst ◽

Michaela Waibel ◽

...

Keyword(s):

Cell Death ◽

Dendritic Cells ◽

Dendritic Cell ◽

Yersinia Enterocolitica ◽

Initial Period ◽

Caspase 8 ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling

ABSTRACT In an initial period (≤4 h) Toll-like receptor 4 (TLR4) signaling is required for Yersinia enterocolitica YopP-induced dendritic cell (DC) death. Later (>4 h), DC die independent of TLR4 signaling. In TLR4-deficient DC caspase 8 cleavage is delayed, indicating that TLR4 signaling accelerates caspase 8 activation, leading to DC death.

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Ubiquitination of ECSIT is crucial for the activation of p65/p50 NF-κBs in Toll-like receptor 4 signaling

Molecular Biology of the Cell ◽

10.1091/mbc.e14-08-1277 ◽

2015 ◽

Vol 26 (1) ◽

pp. 151-160 ◽

Cited By ~ 12

Author(s):

Sae Mi Wi ◽

Jeongho Park ◽

Jae-Hyuck Shim ◽

Eunyoung Chun ◽

Ki-Young Lee

Keyword(s):

Tumor Necrosis ◽

Tumor Necrosis Factor Receptor ◽

Toll Like Receptor ◽

Wild Type ◽

Tlr Signaling ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling ◽

Evolutionarily Conserved ◽

Biochemical Studies ◽

Necrosis Factor

Recent evidence shows that evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) interacts with tumor necrosis factor receptor–associated factor 6 (TRAF6), is ubiquitinated, and contributes to bactericidal activity during Toll-like receptor (TLR) signaling. Here we report a new regulatory role for ECSIT in TLR4 signaling. On TLR4 stimulation, endogenous ECSIT formed a molecular complex with p65/p50 NF-κB proteins. Our biochemical studies showed that ECSIT specifically interacted with p65/p50 NF-κB proteins, which colocalized in the nucleus. Of interest, these effects were critically dependent on ubiquitination of the ECSIT lysine (K) 372 residue. K372A mutant ECSIT did not interact with p65/p50 NF-κB proteins and markedly attenuated nuclear colocalization. In addition, ECSIT-knockdown THP-1 cells could not activate NF-κB DNA-binding activities of p65 and p50, production of proinflammatory cytokines, or NF-κB–dependent gene expression in response to TLR4 stimulation. However, these activities were markedly restored by expressing the wild-type ECSIT protein but not the K372A mutant ECSIT protein. These data strongly suggest that the ubiquitination of ECSIT might have a role in the regulation of NF-κB activity in TLR4 signaling.

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Absence of toll-like receptor 4 (TLR4) signaling in the donor organ reduces ischemia and reperfusion injury in a murine liver transplantation model

Liver Transplantation ◽

10.1002/lt.21251 ◽

2007 ◽

Vol 13 (10) ◽

pp. 1435-1443 ◽

Cited By ~ 80

Author(s):

Xiu-Da Shen ◽

Bibo Ke ◽

Yuan Zhai ◽

Feng Gao ◽

Sei-Ichiro Tsuchihashi ◽

...

Keyword(s):

Liver Transplantation ◽

Reperfusion Injury ◽

Ischemia And Reperfusion ◽

Toll Like Receptor ◽

Donor Organ ◽

Ischemia And Reperfusion Injury ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling ◽

Murine Liver ◽

Transplantation Model

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FTO (Fat-Mass and Obesity-Associated Protein) Participates in Hemorrhage-Induced Thalamic Pain by Stabilizing Toll-Like Receptor 4 Expression in Thalamic Neurons

Stroke ◽

10.1161/strokeaha.121.034173 ◽

2021 ◽

Author(s):

Ganglan Fu ◽

Shibin Du ◽

Tianfeng Huang ◽

Minghui Cao ◽

Xiaozhou Feng ◽

...

Keyword(s):

Fat Mass ◽

Rna Binding ◽

Intraperitoneal Administration ◽

Toll Like Receptor ◽

Tlr4 Expression ◽

Toll Like Receptor 4 ◽

Thalamic Neurons ◽

Adeno Associated Virus ◽

Meclofenamic Acid ◽

Thalamic Pain

Background and Purpose: Hemorrhage-caused gene changes in the thalamus likely contribute to thalamic pain genesis. RNA N 6 -methyladenosine modification is an additional layer of gene regulation. Whether FTO (fat-mass and obesity-associated protein), an N 6 -methyladenosine demethylase, participates in hemorrhage-induced thalamic pain is unknown. Methods: Expression of Fto mRNA and protein was assessed in mouse thalamus after hemorrhage caused by microinjection of Coll IV (type IV collagenase) into unilateral thalamus. Effect of intraperitoneal administration of meclofenamic acid (a FTO inhibitor) or microinjection of adeno-associated virus 5 (AAV5) expressing Cre into the thalamus of Fto fl/fl mice on the Coll IV microinjection–induced TLR4 (Toll-like receptor 4) upregulation and nociceptive hypersensitivity was examined. Effect of thalamic microinjection of AAV5 expressing Fto (AAV5- Fto ) on basal thalamic TLR4 expression and nociceptive thresholds was also analyzed. Additionally, level of N 6 -methyladenosine in Tlr4 mRNA and its binding to FTO or YTHDF2 (YTH N 6 -methyladenosine RNA binding protein 2) were observed. Results: FTO was detected in neuronal nuclei of thalamus. Level of FTO protein, but not mRNA, was time-dependently increased in the ipsilateral thalamus on days 1 to 14 after Coll IV microinjection. Intraperitoneal injection of meclofenamic acid or adeno-associated virus-5 expressing Cre microinjection into Fto fl/fl mouse thalamus attenuated the Coll IV microinjection–induced TLR4 upregulation and tissue damage in the ipsilateral thalamus and development and maintenance of nociceptive hypersensitivities on the contralateral side. Thalamic microinjection of AAV5- Fto increased TLR4 expression and elicited hypersensitivities to mechanical, heat and cold stimuli. Mechanistically, Coll IV microinjection produced an increase in FTO binding to Tlr4 mRNA, an FTO-dependent loss of N 6 -methyladenosine sites in Tlr4 mRNA and a reduction in the binding of YTHDF2 to Tlr4 mRNA in the ipsilateral thalamus. Conclusions: Our findings suggest that FTO participates in hemorrhage-induced thalamic pain by stabilizing TLR4 upregulation in thalamic neurons. FTO may be a potential target for the treatment of this disorder.

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Toll-Like Receptor 4 Signaling and Drug Addiction

Frontiers in Pharmacology ◽

10.3389/fphar.2020.603445 ◽

2020 ◽

Vol 11 ◽

Author(s):

Ruyan Wu ◽

Jun-Xu Li

Keyword(s):

Drug Addiction ◽

Multiple Drug ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling ◽

Drug Classes ◽

Downstream Effectors ◽

Potential Efficacy ◽

Proinflammatory Responses

The emphasis of neuronal alterations and adaptations have long been the main focus of the studies of the mechanistic underpinnings of drug addiction. Recent studies have begun to appreciate the role of innate immune system, especially toll-like receptor 4 (TLR4) signaling in drug reward-associated behaviors and physiology. Drugs like opioids, alcohol and psychostimulants activate TLR4 signaling and subsequently induce proinflammatory responses, which in turn contributes to the development of drug addiction. Inhibition of TLR4 or its downstream effectors attenuated the reinforcing effects of opioids, alcohol and psychostimulants, and this effect is also involved in the withdrawal and relapse-like behaviors of different drug classes. However, conflicting results also argue that TLR4-related immune response may play a minimal part in drug addiction. This review discussed the preclinical evidence that whether TLR4 signaling is involved in multiple drug classes action and the possible mechanisms underlying this effect. Moreover, clinical studies which examined the potential efficacy of immune-base pharmacotherapies in treating drug addiction are also discussed.

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Key Player in Cardiac Hypertrophy, Emphasizing the Role of Toll-Like Receptor 4

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2020.579036 ◽

2020 ◽

Vol 7 ◽

Author(s):

Zheng Xiao ◽

Bin Kong ◽

Hongjie Yang ◽

Chang Dai ◽

Jin Fang ◽

...

Keyword(s):

Inflammatory Response ◽

Cardiac Hypertrophy ◽

Intracellular Signaling ◽

Molecular Mechanisms ◽

New Technologies ◽

Immune Cell ◽

Current Knowledge ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Tlr4 Signaling

Toll-like receptor 4 (TLR4), a key pattern recognition receptor, initiates the innate immune response and leads to chronic and acute inflammation. In the past decades, accumulating evidence has implicated TLR4-mediated inflammatory response in regulation of myocardium hypertrophic remodeling, indicating that regulation of the TLR4 signaling pathway may be an effective strategy for managing cardiac hypertrophy's pathophysiology. Given TLR4's significance, it is imperative to review the molecular mechanisms and roles underlying TLR4 signaling in cardiac hypertrophy. Here, we comprehensively review the current knowledge of TLR4-mediated inflammatory response and its interaction ligands and co-receptors, as well as activation of various intracellular signaling. We also describe the associated roles in promoting immune cell infiltration and inflammatory mediator secretion, that ultimately cause cardiac hypertrophy. Finally, we provide examples of some of the most promising drugs and new technologies that have the potential to attenuate TLR4-mediated inflammatory response and prevent or reverse the ominous cardiac hypertrophy outcomes.

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