TAK1 Is Required for Survival of Mouse Fibroblasts Treated with TRAIL,and Does So by NF-κB Dependent Induction of cFLIPL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a “death ligand”—a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-κB and JNK signalling pathways. To determine the role of TGF-β-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1−/− MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-κB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-κB, protected TAK1−/− MEFs against TRAIL killing, suggesting that TAK1 activation of NF-κB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-κB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1−/− MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1–NF-κB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.     

TGFβ-activated Kinase 1 (TAK1) Inhibition by 5Z-7-Oxozeaenol Attenuates Early Brain Injury after Experimental Subarachnoid Hemorrhage

Accumulating evidence suggests that activation of mitogen-activated protein kinases (MAPKs) and nuclear factor NF-κB exacerbates early brain injury (EBI) following subarachnoid hemorrhage (SAH) by provoking proapoptotic and proinflammatory cellular signaling. Here we evaluate the role of TGFβ-activated kinase 1 (TAK1), a critical regulator of the NF-κB and MAPK pathways, in early brain injury following SAH. Although the expression level of TAK1 did not present significant alternation in the basal temporal lobe after SAH, the expression of phosphorylated TAK1 (Thr-187, p-TAK1) showed a substantial increase 24 h post-SAH. Intracerebroventricular injection of a selective TAK1 inhibitor (10 min post-SAH), 5Z-7-oxozeaenol (OZ), significantly reduced the levels of TAK1 and p-TAK1 at 24 h post-SAH. Involvement of MAPKs and NF-κB signaling pathways was revealed that OZ inhibited SAH-induced phosphorylation of p38 and JNK, the nuclear translocation of NF-κB p65, and degradation of IκBα. Furthermore, OZ administration diminished the SAH-induced apoptosis and EBI. As a result, neurological deficits caused by SAH were reversed. Our findings suggest that TAK1 inhibition confers marked neuroprotection against EBI following SAH. Therefore, TAK1 might be a promising new molecular target for the treatment of SAH.     

Pellino-1 Positively Regulates Toll-like Receptor (TLR) 2 and TLR4 Signaling and Is Suppressed upon Induction of Endotoxin Tolerance

Endotoxin tolerance reprograms Toll-like receptor (TLR) 4-mediated macrophage responses by attenuating induction of proinflammatory cytokines while retaining expression of anti-inflammatory and antimicrobial mediators. We previously demonstrated deficient TLR4-induced activation of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, and TANK-binding kinase (TBK) 1 as critical hallmarks of endotoxin tolerance, but mechanisms remain unclear. In this study, we examined the role of the E3 ubiquitin ligase Pellino-1 in endotoxin tolerance and TLR signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in macrophages from mice injected with saline and in medium-pretreated human monocytes, THP-1, and MonoMac-6 cells, whereas endotoxin tolerization abrogated LPS inducibility of Pellino-1. Overexpression of Pellino-1 in 293/TLR2 and 293/TLR4/MD2 cells enhanced TLR2- and TLR4-induced nuclear factor κB (NF-κB) and expression of IL-8 mRNA, whereas Pellino-1 knockdown reduced these responses. Pellino-1 ablation in THP-1 cells impaired induction of myeloid differentiation primary response protein (MyD88), and Toll-IL-1R domain-containing adapter inducing IFN-β (TRIF)-dependent cytokine genes in response to TLR4 and TLR2 agonists and heat-killed Escherichia coli and Staphylococcus aureus, whereas only weakly affecting phagocytosis of heat-killed bacteria. Co-expressed Pellino-1 potentiated NF-κB activation driven by transfected MyD88, TRIF, IRAK1, TBK1, TGF-β-activated kinase (TAK) 1, and TNFR-associated factor 6, whereas not affecting p65-induced responses. Mechanistically, Pellino-1 increased LPS-driven K63-linked polyubiquitination of IRAK1, TBK1, TAK1, and phosphorylation of TBK1 and IFN regulatory factor 3. These results reveal a novel mechanism by which endotoxin tolerance re-programs TLR4 signaling via suppression of Pellino-1, a positive regulator of MyD88- and TRIF-dependent signaling that promotes K63-linked polyubiquitination of IRAK1, TBK1, and TAK1.     

Liver Kinase B1 Suppresses Lipopolysaccharide-induced Nuclear Factor κB (NF-κB) Activation in Macrophages

Liver kinase B1 (LKB1), a serine/threonine kinase, is a tumor suppressor and metabolic regulator. Recent data suggest that LKB1 is essential in regulating homeostasis of hematopoietic cells and immune responses. However, its role in macrophages and innate immune system remains unclear. Here we report that macrophage LKB1 inhibits pro-inflammatory signaling in response to LPS. LPS-induced pro-inflammatory cytokines and pro-inflammatory enzymes were monitored in bone marrow-derived macrophages isolated from myeloid cell-specific LKB1 knock out mice and their wild type littermate control mice. LPS induced higher levels of pro-inflammatory cytokines and pro-inflammatory enzymes in bone marrow-derived macrophages from LKB1 KO than those from wild type mice. Consistently, LPS induced higher levels of NF-κB activation in LKB1-deficient macrophages than those in wild type. Further, LPS stimulation significantly increased LKB1 phosphorylation at serine 428, which promoted its binding to IκB kinaseβ (IKKβ), resulting in the inhibition of NF-κB. Finally, LPS injection caused higher levels of cytokine release and more severe tissue injury in the lung tissues of LKB1 KO mice than in those of control mice. We conclude that LKB1 inhibits LPS-induced NF-κB activation in macrophages.     

TAK1-ECSIT-TRAF6 Complex Plays a Key Role in the TLR4 Signal to Activate NF-κB

ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) is known as a multifunctional regulator in different signals, including Toll-like receptors (TLRs), TGF-β, and BMP. Here, we report a new regulatory role of ECSIT in TLR4-mediated signal. By LPS stimulation, ECSIT formed a high molecular endogenous complex including TAK1 and TRAF6, in which ECSIT interacted with each protein and regulated TAK1 activity, leading to the activation of NF-κB. ECSIT-knockdown THP-1 (ECSIT^KD THP-1) cells exhibited severe impairments in NF-κB activity, cytokine production, and NF-κB-dependent gene expression, whereas those were dramatically restored by reintroduction of wild type (WT) ECSIT gene. Interestingly, ECSIT mutants, which lack a specific interacting domain for either TAK1 or TRAF6, could not restore these activities. Moreover, no significant changes in both NF-κB activity and cytokine production induced by TLR4 could be seen in TAK1^KD or TRAF6^KD THP-1 cells transduced by WT ECSIT, strongly suggesting the essential requirement of TAK1-ECSIT-TRAF6 complex in TLR4 signaling. Taken together, our data demonstrate that the ECSIT complex, including TAK1 and TRAF6, plays a pivotal role in TLR4-mediated signals to activate NF-κB.     

MEKK3 is required for lysophosphatidic acid-induced NF-κB activation

Lysophosphatidic acid (LPA) is a potent agonist that exerts various cellular functions on many cell types through binding to its cognate G protein-coupled receptors (GPCRs). Although LPA induces NF-κB activation by acting on its GPCR receptor, the molecular mechanism of LPA receptor-mediated NF-κB activation remains to be well defined. In the present study, by using MEKK3-, TAK1-, and IKKβ-deficient murine embryonic fibroblasts (MEFs), we found that MEKK3 but not TAK1 deficiency impairs LPA and protein kinase C (PKC)-induced IκB kinase (IKK)-NF-κB activation, and IKKβ is required for PKC-induced NF-κB activation. In addition, we demonstrate that LPA and PKC-induced IL-6 and MIP-2 production are abolished in the absence of MEKK3 but not TAK1. Together, our results provide the genetic evidence that MEKK3 but not TAK1 is required for LPA receptor-mediated IKK-NF-κB activation.     

The Therapeutic Potential and Mechanisms of Action of Quercetin in Relation to Lipopolysaccharide-Induced Sepsis In Vitro and In Vivo

Sepsis caused by Gram-negative bacterial infection is characterized by extensive inflammatory cytokine production, which leads to multiple organ failure and a high lethality rate. Therefore, compounds that are able to alleviate profound inflammatory responses may have therapeutic potential in relation to sepsis. Quercetin, one of the flavonoids found widely in the human diet, has been reported to have many health benefits, but the mechanisms underlying its biological effects remain obscure. In the present study, our aim was to investigate the molecular mechanisms by which quercetin inhibits lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and to evaluate the capacity of quercetin to attenuate the mortality rate in a mice model of lethal sepsis. Our results show that quercetin significantly attenuates LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 macrophages. The LPS-stimulated phosphorylations of the inhibitors of κB kinase (IKKs), Akt, and c-Jun N-terminal kinase (JNK) are also inhibited by quercetin. Quercetin causes a significant reduction in the phosphorylation and degradation of inhibitor of κBα (IκBα) and in the nuclear level of nuclear factor-κB (NF-κB), the latter being associated with decreased NF-κB binding activity. Most importantly, acute administration of quercetin reduces the lethality rate and circulating levels of TNF-α and IL-1β in C57BL/6J mice with endotoxemia induced by LPS, whereas chronic dietary supplementation with quercetin shows no inhibitory effect on serum TNF-α and IL-1β levels. These findings provide clues that quercetin may be a promising agent for the prevention of systemic inflammatory diseases such as sepsis.     

Role of TLR4/NF-κB in Damage to Intestinal Mucosa Barrier Function and Bacterial Translocation in Rats Exposed to Hypoxia

The role of Toll-like receptor 4 (TLR4)/nuclear factor-kappa-B (NF-κB) in intestinal mucosal barrier damage and bacterial translocation under hypoxic exposure is unclear. Here, we investigated their role using an acute hypobaric hypoxia model. Adult Sprague-Dawley rats were divided into control (C), hypoxia (H), hypoxia+NF-κB inhibitor pyrrolidinedithiocarbamic acid (PDTC) (100 mg. kg) (HP), hypoxia+0.5 mg/kg lipopolysaccharide (HPL), and hypoxia+PDTC+LPS (HPL) group. Except control group, other four groups were placed in a hypobaric chamber set at 7000 m. Samples were collected at 72 h after pressure reduction. Damage in ultrastructure of the intestinal tract was examined by transmission electron microscopy and bacterial translocation was detected by cultivation. Kinetic turbidimetric assay was used to measure the serum LPS.ELISA was performed to detect TNF-α and IL-6 serum concentrations. Fluorescent quantitative RT-PCR was used to measure TLR4 mRNA levels was measured using quantitative RT-PCR and protein of NF-κB p65 was measured by western blotting. Different degrees of intestinal mucosa damage were observed in groups H and HL. The damage was significantly alleviated after blockage of the TLR4/NF-κB signaling pathway. PDTC- treatment also reversed hyoxia- and LPS-induced bacterial translocation rate and increased serum levels of LPS, TNF-α, and IL-6. TLR4 mRNA levels and NF-κB p65 expression were consistent with the serum factor results. This study suggested that TLR4 and NF-κB expression increased in rat intestinal tissues after acute hypoxia exposure. PDTC-treatment reversed TLR4 and NF-κB upregulation and alleviated damage to the intestinal tract and bacterial translocation. Thus, the TLR4/NF-κB signaling pathway may be critical to the mechanism underlying hypoxia-induced damage to intestinal barrier function and bacterial translocation.     

Anisomycin protects against sepsis by attenuating IκB kinase-dependent NF-κB activation and inflammatory gene expression

Anisomycin is known to inhibit eukaryotic protein synthesis and has been established as an antibiotic and anticancer drug. However, the molecular targets of anisomycin and its mechanism of action have not been explained in macrophages. Here, we demonstrated the anti-inflammatory effects of anisomycin both in vivo and in vitro. We found that anisomycin decreased the mortality rate of macrophages in cecal ligation and puncture (CLP)- and lipopolysaccharide (LPS)-induced acute sepsis. It also declined the gene expression of proinflammatory mediators such as inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β as well as the nitric oxide and proinflammatory cytokines production in macrophages subjected to LPS-induced acute sepsis. Furthermore, anisomycin attenuated nuclear factor (NF)-κB activation in LPS-induced macrophages, which correlated with the inhibition of phosphorylation of NF-κB-inducing kinase and IκB kinase, phosphorylation and IκBα proteolytic degradation, and NF-κB p65 subunit nuclear translocation. These results suggest that anisomycin prevented acute inflammation by inhibiting NF-κB-related inflammatory gene expression and could be a potential therapeutic candidate for sepsis.