Overexpression Of TBK1 Research Articles

Cardiomyocyte-specific Tbk1 deletion aggravated chronic doxorubicin cardiotoxicity via inhibition of mitophagy

Doxorubicin (Dox) use is limited by Dox-induced cardiotoxicity. TANK-blinding kinase 1 (TBK1) is an important kinase involved in the regulation of mitophagy, but the role of TBK1 in cardiomyocytes in chronic Dox-induced cardiomyopathy remains unclear. Cardiomyocyte-specific Tbk1 knockout (Tbk1CKO) mice received Dox (6 mg/kg, injected intraperitoneally) once a week for 4 times, and cardiac assessment was performed 4 weeks after the final Dox injection. Adenoviruses encoding Tbk1 or containing shRNA targeting Tbk1, or a TBK1 phosphorylation inhibitor were used for overexpression or knockdown of Tbk1, or inhibit phosphorylation of TBK1 in isolated primary cardiomyocytes. Our results revealed that moderate Dox challenge decreased TBK1 phosphorylation (with no effect on TBK1 protein levels), resulting in compromised myocardial function, obvious mortality and overt interstitial fibrosis, and the effects were accentuated by Tbk1 deletion. Dox provoked mitochondrial membrane potential collapse and oxidative stress, the effects of which were exacerbated and mitigated by Tbk1 knockdown, specific inhibition of phosphorylation and overexpression, respectively. However, Tbk1 （Ser172A） overexpression did not alleviate these effects. Further scrutiny revealed that TBK1 exerted protective effects on mitochondria via SQSTM1/P62-mediated mitophagy. Tbk1 overexpression mediated cardioprotective effects on Dox-induced cardiotoxicity were cancelled off by Sqstm1/P62 knockdown. Moreover, TBK1-mitophagy-mitochondria cascade was confirmed in heart tissues from dilated cardiomyopathy patients. Taken together, our findings denoted a pivotal role of TBK1 in Dox-induced mitochondrial injury and cardiotoxicity possibly through its phosphorylation and SQSTM1/P62-mediated mitophagy.

Identification of multifunctionality of grass carp (Ctenopharyngodon idella) TBK1 during bacterial infection

TBK1 is an atypical IκB kinase family member with a set of functions. It is involved in congenital immunization and autophagy in mammals. In this study, we reported that grass carp TBK1 gene expression could be upregulated by bacterial infection. Overexpression of TBK1 could decrease the number of adhesive bacteria in CIK cells. TBK1 could promote cellular migration, proliferation, vitality, and anti-apoptosis ability. Furthermore, the expression of TBK1 could activate the NF-κB signaling pathway by inducing inflammatory cytokines. In addition, we found that the grass carp TBK1 could cause the autophagy level of CIK cells within the decreasing level of p62 protein. Our finding indicated that TBK1 participated in grass carp innate immune progress and autophagy. This study provides evidence of the positive regulation of TBK1 in teleost innate immunity with its multiple functions. It thus may provide important information about the defense and immune mechanisms used by teleost against pathogens.

Caragana rosea Turcz Methanol Extract Inhibits Lipopolysaccharide-Induced Inflammatory Responses by Suppressing the TLR4/NF-κB/IRF3 Signaling Pathways.

Caragana rosea Turcz, which belongs to the Leguminosae family, is a small shrub found in Northern and Eastern China that is known to possess anti-inflammatory properties and is used to treat fever, asthma, and cough. However, the underlying molecular mechanisms of its anti-inflammatory effects are unknown. Therefore, we used lipopolysaccharide (LPS) in RAW264.7 macrophages to investigate the molecular mechanisms that underlie the anti-inflammatory activities of a methanol extract of Caragana rosea (Cr-ME). We showed that Cr-ME reduced the production of nitric oxide (NO) and mRNA levels of iNOS, TNF-α, and IL-6 in a concentration-dependent manner. We also found that Cr-ME blocked MyD88- and TBK1-induced NF-κB and IRF3 promoter activity, suggesting that it affects multiple targets. Moreover, Cr-ME reduced the phosphorylation levels of IκBα, IKKα/β and IRF3 in a time-dependent manner and regulated the upstream NF-κB proteins Syk and Src, and the IRF3 protein TBK1. Upon overexpression of Src and TBK1, Cr-ME stimulation attenuated the phosphorylation of the NF-κB subunits p50 and p65 and IRF3 signaling. Together, our results suggest that the anti-inflammatory activity of Cr-ME occurs by inhibiting the NF-κB and IRF3 signaling pathways.

Parkin overexpression alleviates cardiac aging through facilitating K63-polyubiquitination of TBK1 to facilitate mitophagy

Cumulative clinical and experimental evidence has revealed a cardinal role for mitochondrial integrity in cardiac aging. Parkin-mediated mitophagy is essential to ensure mitochondrial quality control in myocardium. This study was designed to examine the impact of Parkin overexpression on aging-induced myocardial anomalies and the underlying mechanisms with a focus on Parkin-regulated mitophagy. Cardiac function, myocardial apoptosis, mitochondrial ultrastructure and mitophagy were examined in young (3 mo) and old (24–26 mo) wild-type (WT) and Parkin transgenic mice. Our data revealed compromised myocardial function and mitochondrial morphology along with overtly apoptosis with advanced aging, the effects of which were attenuated by Parkin overexpression. Advanced aging dampened mitophagy as evidenced by decreased levels of Parkin, LC3II, phosphorylation of p62 and TBK1 in isolated mitochondria as well as reduced mitochondria autophagosomes, the effects of which were mitigated by restoration of mitophagy via Parkin overexpression. Using the low-dose doxorubicin (DOX) in vitro model of cell senescence, we noted that Parkin-offered beneficial effect against senescence was abolished by the TBK1 kinase inhibitor BX795. With TBK1 overexpression in cardiomyocytes, we uncovered the interaction of Parkin with TBK1 using a Co-immunoprecipitation (Co-IP) assay. The interaction of Parkin with TBK1 contributed to K63-linked polyubiquitination of TBK1. Our study also noted that DOX disturbed K63-linked polyubiquitination of TBK1 with downregulation of Parkin. Parkin overexpression promoted K63-linked polyubiquitination of TBK1 through Lys30 and Lys401 residues to foster TBK1 phosphorylation to facilitate efficient mitophagy. In summary, these findings suggested that Parkin effectively rescued cardiac aging through promoting K63-linked polyubiquitination of TBK1 to facilitate mitophagy.

Identification of prognostic biomarkers and correlations with immune infiltrates among cGAS-STING in hepatocellular carcinoma.

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway induces innate immunity by activating the production of inflammatory cytokines and type I interferons. Recently, studies revealed that self-DNA from by-products of chromosome instability and tumors could activate the cGAS-STING pathway, and subsequently promote or inhibit tumor development. However, the prognostic value and correlations with immune infiltrates of the cGAS-STING pathway in hepatocellular carcinoma (HCC) have not been clarified. In the present study, we used the Molecular Signatures Database, Oncomine, UALCAN, Human Protein Atlas, Kaplan–Meier plotter, LinkedOmics, and Tumor Immune Estimation Resource databases. Overexpression of XRCC5, IRF3, TRIM21, STAT6, DDX41, TBK1, XRCC6, TREX1, PRKDC, and TMEM173 was markedly correlated with clinical stages and pathological grades in HCC. Moreover, higher mRNA expression of XRCC5, XRCC6, and PRKDC was significantly related with shorter overall survival. However, higher mRNA expression of IFI16, STAT6, NLRC3, and TMEM173 was associated with favorable overall survival. Our results suggested that the kinase targets of the cGAS-STING pathway included the SRC family of tyrosine kinases (LCK and LYN), phosphoinositide 3-kinase-related protein kinase (PIKK) family kinases (ATM and ATR), and mitogen-activated protein kinase 1 (MAPK1). Furthermore, we identified significant correlations among the expression of cGAS-STING pathway and infiltration of B cells, CD4+T cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells in HCC. The expression of the cGAS-STING pathway also exhibited strong relationships with diverse immune marker sets in HCC. These findings suggest that cGAS-STING pathway members may be used as prognostic biomarkers and immunotherapeutic targets HCC patients.

Deletion of Tbk1 disrupts autophagy and reproduces behavioral and locomotor symptoms of FTD-ALS in mice

Haploinsufficiency of the protein kinase Tbk1 has shown to cause both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, the pathogenic mechanisms are unclear. Here we show that conditional neuronal deletion of Tbk1 in leads to cognitive and locomotor deficits in mice. Tbk1-NKO mice exhibited numerous neuropathological changes, including neurofibrillary tangles, abnormal dendrites, reduced dendritic spine density, and cortical synapse loss. The Purkinje cell layer of the cerebellum presented dendritic swelling, abnormally shaped astrocytes, and p62- and ubiquitin-positive aggregates, suggesting impaired autophagy. Inhibition of autophagic flux with bafilomycin A increased total Tkb1 levels in motor neuron-like cells in vitro, suggesting autophagy-dependent degradation of Tbk1. Although Tbk1 over-expression did not affect mutant SOD1 levels in SOD1G93A-transfected cells, it increased the soluble/insoluble ratio and reduced the number and size of SOD1G93A aggregates. Finally, in vivo experiments showed that Tkb1 expression was reduced in SOD1G93A ALS transgenic mice, which showed decreased p62 protein aggregation and extended survival after ICV injection of adeno-associated viral vectors encoding Tbk1. These data shed light on the neuropathological changes that result from Tbk1 deficiency and hint at impaired autophagy as a contributing factor to the cognitive and locomotor deficits that characterize FTD-ALS in patients with Tkb1 haploinsufficiency.

BinCARD2 as a positive regulator of interferon response in innate immunity

Innate immunity is a system that recognizes primarily and excludes pathogenic microorganism. MAVS/IPS-1/Cardif/Visa functions as an adapter protein for RIG-I like receptors (RLRs) and plays a key role in the production of antiviral proteins, interferons (IFNs), for RNA viruses. However, the activation mechanism is not fully understood. Here, we show that BinCARD isoform2 (BinCARD2), carrying CARD domain structure like MAVS, functions in innate immune response. Knockdown of BinCARD2 reduced the RLR ligand-induced expression of IFN-β mRNA and activation of the IFNB promoter. The activation of the IFNB promoter by overexpression of MAVS or TBK1 was suppressed by silencing of BinCARD2, but no effect on IFNB promoter activation by overexpression of TRIF or constitutive activated IRF-3. Furthermore, we confirmed that BinCARD2 protein associated with MAVS but not TBK1 by immunoprecipitation and colocalized with MAVS. Accordingly, we investigated whether BinCARD2 was involved in MAVS activation and showed that siBinCARD2 did not affect RIG-I/MAVS binding but impaired the MAVS oligomerization. Moreover, we infected A549 cells with vesicular stomatitis virus (VSV) and found that induction of IFN-β and IL-6 mRNA after VSV infection was decreased by BinCARD2 knockdown. Thus, these data may suggest that BinCARD2 associates with MAVS to positively modulate the oligomerization in the RIG-I like receptors pathway and activates innate immune response.

Andrographolide suppresses TRIF-dependent signaling of toll-like receptors by targeting TBK1

Toll-like receptors (TLRs) play a crucial role in danger recognition and induction of innate immune response against bacterial and viral infections. The TLR adaptor molecule, toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF), facilitates TLR3 and TLR4 signaling, leading to the activation of the transcription factor, NF-κB and interferon regulatory factor 3 (IRF3). Andrographolide, the active component of Andrographis paniculata, exerts anti-inflammatory effects; however, the principal molecular mechanisms remain unclear. The objective of this study was to investigate the role of andrographolide in TLR signaling pathways. Andrographolide suppressed NF-κB activation as well as COX-2 expression induced by TLR3 or TLR4 agonists. Andrographolide also suppressed the activation of IRF3 and the expression of interferon inducible protein-10 (IP-10) induced by TLR3 or TLR4 agonists. Andrographolide attenuated ligand-independent activation of IRF3 following overexpression of TRIF, TBK1, or IRF3. Furthermore, andrographolide inhibited TBK1 kinase activity in vitro. These results indicate that andrographolide modulates the TRIF-dependent pathway of TLRs by targeting TBK1 and represents a potential new anti-inflammatory candidate.

Nontypeable Haemophilus influenzae DNA stimulates type I interferon expression via STING signaling pathway

Nontypeable Haemophilus influenzae (NTHI) is one of the leading causes of acute exacerbations of COPD (AECOPD). Although the immunoregulation function of NTHI outer member protein and endotoxin were confirmed, the role of NTHI DNA in activating immune responses remains to be elucidated. In this study, we found expression of IFN-β and IFN stimulated gene CXCL10 in host cells was forcefully elevated after treating with NTHI and NTHI DNA. Interestingly, we tested increased level of STING in NTHI infected mice lung. Meanwhile, STING expression in lung of mimic COPD murine model was higher than healthy mice after NTHI infection. Importantly, knockout of STING or overexpression of STING, TBK1 and IRF3 respectively impaired or enhanced IFN-β and CXCL10 expression during treating with NTHI and NTHI DNA. NTHI and NTHI DNA-induced I-IFN response appeared to be mediated by cGAS. Collectively, we suggested that NTHI DNA as a PAMP triggered I-IFN response, which was STING/TBK1/IRF3 dependent. SummaryNTHI is the leading cause of acute exacerbations of COPD (AECOPD). Since AECOPD is an immune event, it is meaningful to elucidate the mechanism under NTHI induced immune response. It has been revealed that lipooligosaccharides and protein of NTHI could induce host immune response, but the function of NTHI nuclide acid during infection is unclear. In this research, we demonstrate NTHI DNA is a trigger for I-IFN expression, and the STING/TBK1/IRF3 pathway plays an integral role in sensing NTHI DNA to induce I-IFN expression. Moreover, by long-term intrabronchial infection of LPS, we constructed a mimic COPD murine model, in which the STING expression in lung tissues were higher than healthy mice after NTHI infection, which led us to surmise that NTHI cause AECOPD by inducing I-IFN production via STING signal pathway.

High-throughput microfluidic platform for culture of 3D-kidney tissue models

Toll-like receptors (TLRs) play a crucial role in danger recognition and induction of innate immune response against bacterial and viral infections. The TLR adaptor molecule, toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF), facilitates TLR3 and TLR4 signaling, leading to the activation of the transcription factor, NF-κB and interferon regulatory factor 3 (IRF3). Andrographolide, the active component of Andrographis paniculata, exerts anti-inflammatory effects; however, the principal molecular mechanisms remain unclear. The objective of this study was to investigate the role of andrographolide in TLR signaling pathways. Andrographolide suppressed NF-κB activation as well as COX-2 expression induced by TLR3 or TLR4 agonists. Andrographolide also suppressed the activation of IRF3 and the expression of interferon inducible protein-10 (IP-10) induced by TLR3 or TLR4 agonists. Andrographolide attenuated ligand-independent activation of IRF3 following overexpression of TRIF, TBK1, or IRF3. Furthermore, andrographolide inhibited TBK1 kinase activity in vitro. These results indicate that andrographolide modulates the TRIF-dependent pathway of TLRs by targeting TBK1 and represents a potential new anti-inflammatory candidate.

Role of Tank-Binding Kinase in Neurodegeneration Produced by Spinocerebellar Ataxia Type 13

Inherited mutations in the Kv3.3 potassium channel, such as the G952R Kv3.3 mutation, Kv3.3 mutations result in Spinocerebellar Ataxia 13 (SCA13). In contrast to wild type Kv3.3 channels, G592R mutant channels have slowed inactivation and fail to trigger the formation of a Hax-1- and Arp2/3- dependent actin cytoskeleton under the plasma membrane, but significantly increase the rate of cell death in transfected cells. To determine which transduction pathways are activated by G592R Kv3.3 we carried out an in situ kinase profiling screen to compare levels of activity of protein kinases in the cerebella of G592R-knock-in mice with those of wild type animals. The activity of TBK1 was found to be significantly elevated in the cerebellum but not the cerebral cortex of G592R Kv3.3 mice. This result was confirmed by western blotting. We found that enhanced TBK1 activity accounts for the slowed inactivation of the mutant channel. Pharmacological inhibition of TBK1 increased the rate of inactivation of Kv3.3 currents, while TBK1 overexpression slowed their inactivation rate. In order to explore the role of TBK1 in cell death caused by G592R Kv3.3, we measured levels of cleaved caspase 7 in different conditions, and found that the level of cleaved caspase 7 is increased in cells expressing mutant Kv3.3 channels compared with that in cells expressing wild type channels, particularly under condition of depolarization with high K+. Pretreatment with the TBK1 inhibitor, however, reduced expression levels of cleaved caspase 7 under high K+ stimulation in both types of cells, but not in untransfected cells. Our results suggest that the interactions of the Kv3.3 potassium channel with TBK1 and Hax-1 regulate channel gating and may link the channel to signaling pathways that protect cerebellar neurons from apoptosis.

TBK1 Promote Bladder Cancer Cell Proliferation and Migration via Akt Signaling.

Bladder cancer is a challenging and fatal malignancy and the improvement in prognosis is limited over years. Deep understanding the mechanism of bladder cancer tumorigenesis and progression will help to discover novel and effective treatment strategies. In this study, we identify non-canonical IkB kinase TBK1 is up-regulated in bladder cancer tissue and cell lines. Knockdown of TBK1 markedly inhibits cell proliferation and migration. Inhibition of TBK1 kinase activity by BX795 significantly attenuates bladder cancer cell proliferation and migration. Mechanistic study shows that overexpression of TBK1 promoted the phosphorylation of Akt, whereas knockdown of TBK1 reverses this action. Taken together, our data suggest that TBK1 modulates the malignant behaviors of bladder cancer cell via Akt signaling, revealing new insights in discovering new therapy target for bladder cancer.

CFLIPL Interrupts IRF3-CBP-DNA Interactions To Inhibit IRF3-Driven Transcription.

Type I IFN induction is critical for antiviral and anticancer defenses. Proper downregulation of type I IFN is equally important to avoid deleterious imbalances in the immune response. The cellular FLIP long isoform protein (cFLIPL) controls type I IFN production, but opposing publications show it as either an inhibitor or inducer of type I IFN synthesis. Regardless, the mechanistic basis for cFLIPL regulation is unknown. Because cFLIPL is important in immune cell development and proliferation, and is a target for cancer therapies, it is important to identify how cFLIPL regulates type I IFN production. Data in this study show that cFLIPL inhibits IFN regulatory factor 3 (IRF3), a transcription factor central for IFN-β and IFN-stimulated gene expression. This inhibition occurs during virus infection, cellular exposure to polyinosinic-polycytidylic acid, or TBK1 overexpression. This inhibition is independent of capase-8 activity. cFLIPL binds to IRF3 and disrupts IRF3 interaction with its IFN-β promoter and its coactivator protein (CREB-binding protein). Mutational analyses reveal that cFLIPL nuclear localization is necessary and sufficient for inhibitory function. This suggests that nuclear cFLIPL prevents IRF3 enhanceosome formation. Unlike other cellular IRF3 inhibitors, cFLIPL did not degrade or dephosphorylate IRF3. Thus, cFLIPL represents a different cellular strategy to inhibit type I IFN production. This new cFLIPL function must be considered to accurately understand how cFLIPL affects immune system development and regulation.

A Glaucoma-Associated Variant of Optineurin, M98K, Activates Tbk1 to Enhance Autophagosome Formation and Retinal Cell Death Dependent on Ser177 Phosphorylation of Optineurin.

Certain missense mutations in optineurin/OPTN and amplification of TBK1 are associated with normal tension glaucoma. A glaucoma-associated variant of OPTN, M98K, induces autophagic degradation of transferrin receptor (TFRC) and death in retinal cells. Here, we have explored the role of Tbk1 in M98K-OPTN-induced autophagy and cell death, and the effect of Tbk1 overexpression in retinal cells. Cell death induced by M98K-OPTN was dependent on Tbk1 as seen by the effect of Tbk1 knockdown and blocking of Tbk1 activity by a chemical inhibitor. Inhibition of Tbk1 also restores M98K-OPTN-induced transferrin receptor degradation. M98K-OPTN-induced autophagosome formation, autophagy and cell death were dependent on its phosphorylation at S177 by Tbk1. Knockdown of OPTN reduced starvation-induced autophagosome formation. M98K-OPTN expressing cells showed higher levels of Tbk1 activation and enhanced phosphorylation at Ser177 compared to WT-OPTN expressing cells. M98K-OPTN-induced activation of Tbk1 and its ability to be phosphorylated better by Tbk1 was dependent on ubiquitin binding. Phosphorylated M98K-OPTN localized specifically to autophagosomes and endogenous Tbk1 showed increased localization to autophagosomes in M98K-OPTN expressing cells. Overexpression of Tbk1 induced cell death and caspase-3 activation that were dependent on its catalytic activity. Tbk1-induced cell death possibly involves autophagy, as shown by the effect of Atg5 knockdown, and requirement of autophagic function of OPTN. Our results show that phosphorylation of Ser177 plays a crucial role in M98K-OPTN-induced autophagosome formation, autophagy flux and retinal cell death. In addition, we provide evidence for cross talk between two glaucoma associated proteins and their inter-dependence to mediate autophagy-dependent cell death.

Fructose-1,6-bisphosphate suppresses lipopolysaccharide-induced expression of ICAM-1 through modulation of toll-like receptor-4 signaling in brain endothelial cells.

Fructose-1,6-bisphosphate (FBP) is a glycolytic intermediate with salutary effects in various brain injury models, but its neuroprotective mechanism is incompletely understood. In this study, we examined the effects of FBP on the expression of adhesion molecules in cerebrovascular endothelial cells and explored the possible mechanisms therein involved. FBP significantly down-regulated lipopolysaccharide (LPS)-induced expression of adhesion molecules and leukocyte adhesion to brain endothelial cells and inhibited NF-κB activity, which is implicated in the expression of adhesion molecules. FBP abrogated ICAM-1 expression and NF-κB activation induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or overexpression of MyD88 or TRAF6. FBP suppressed TRAF6-induced phosphorylation of TAK1, IKKβ and IκBα, but fail to affect NF-κB activity induced by ectopic expression of IKKβ. In addition, LPS-induced IRAK-1 phosphorylation was inhibited by FBP, suggesting the presence of multiple molecular targets of FBP in MyD88-dependent signaling pathway. FBP significantly attenuated ICAM-1 expression and NF-κB activity induced by poly[I:C] or overexpression of TRIF or TBK1. FBP significantly repressed the expression of interferon-β (IFN-β) and the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C] or overexpression of TRIF or TBK1, but fail to affect IRF3 activity induced by ectopic expression of constitutively active IRF3. Overall, our results demonstrate that FBP modulates both MyD88- and TRIF-dependent signaling pathways of TLR4 and subsequent inflammatory responses in brain endothelial cells, providing insight into its neuroprotective mechanism in brain injury associated with inflammation.

Gambogic Acid Disrupts Toll-like Receptor4 Activation by Blocking Lipopolysaccharides Binding to Myeloid Differentiation Factor 2.

Our body’s immune system has defense mechanisms against pathogens such as viruses and bacteria. Immune responses are primarily initiated by the activation of toll-like receptors (TLRs). In particular, TLR4 is well-characterized and is known to be activated by gram-negative bacteria and tissue damage signals. TLR4 requires myeloid differentiation factor 2 (MD2) as a co-receptor to recognize its ligand, lipopolysaccharides (LPS), which is an extracellular membrane component of gram-negative bacteria. Gambogic acid is a xanthonoid isolated from brownish or orange resin extracted from Garcinia hanburyi. Its primary effect is tumor suppression. Since inflammatory responses are related to the development of cancer, we hypothesized that gambogic acid may regulate TLR4 activation. Our results demonstrated that gambogic acid decreased the expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-12, and IL-1β) in both mRNA and protein levels in bone marrow-derived primary macrophages after stimulation with LPS. Gambogic acid did not inhibit the activation of Interferon regulatory factor 3 (IRF3) induced by TBK1 overexpression in a luciferase reporter gene assay using IFN-β-PRD III-I-luc. An in vitro kinase assay using recombinant TBK1 revealed that gambogic acid did not directly inhibit TBK1 kinase activity, and instead suppressed the binding of LPS to MD2, as determined by an in vitro binding assay and confocal microscopy analysis. Together, our results demonstrate that gambogic acid disrupts LPS interaction with the TLR4/MD2 complex, the novel mechanism by which it suppresses TLR4 activation.

MiR-429 Determines Poor Outcome and Inhibits Pancreatic Ductal Adenocarcinoma Growth by Targeting TBK1

Background: Pancreatic ductal adenocarcinoma (PDAC) ranks fourth on the list of cancer-related causes of death and its prognosis has not improved significantly over the past decades. Deregulation or dysfunction of miRNAs contribute to cancer development. Previous data indicates that miR-429 is involved in the pathogenesis of PDAC. However, the role of miR-429 in PDAC remained unknown. Methods: MiR-429 levels in sample tissues of 78 patients and in PANC1 and SW1990 cell lines were quantified by real-time PCR. MiR-429 expression was modulated using specific pre- and anti-miRNAs and cell growth was assayed by MTT analysis. Bioinformatics prediction of the miR-429 putative target genes was performed and luciferase assays confirmed TBK1 as a direct target gene. TBK1 levels in PDAC tissues were analyzed by immunohistochemistry. Results: MiR-429 was remarkably decreased in PDAC tissues and cell lines. Lower miR-429 expression in PDAC tissues significantly correlated with shorter survival of PDAC patients. Overexpression of miR-429 inhibited PDAC cell lines growth in vitro and vice versa. TBK1 was found to be the direct target gene of miR-429. Higher TBK1 protein level in PDAC tissues correlated with shorter survival of PDAC patients. Overexpression of TBK1 partly restored cell proliferation. Conclusions: Low level of miR-429 and high level of TBK1 in PDAC promoted PDAC cells growth which might be related to the low survival rate of PDAC patients. MiR-429 play its role in PDAC by targeting TBK1.

DOK3 is required for IFN-β production by enabling TRAF3/TBK1 complex formation and IRF3 activation.

The downstream of kinase (DOK) family of adaptors is generally involved in the negative regulation of signaling pathways. DOK1, 2, and 3 were shown to attenuate TLR4 signaling by inhibiting Ras-ERK activation. In this study, we elucidated a novel role for DOK3 in IFN-β production. Macrophages lacking DOK3 were impaired in IFN-β synthesis upon influenza virus infection or polyinosinic-polyribocytidylic acid stimulation. In the absence of DOK3, the transcription factor IFN regulatory factor 3 was not phosphorylated and could not translocate to the nucleus to activate ifn-β gene expression. Interestingly, polyinosinic-polyribocytidylic acid-induced formation of the upstream TNFR-associated factor (TRAF) 3/TANK-binding kinase (TBK) 1 complex was compromised in dok3(-/-) macrophages. DOK3 was shown to bind TBK1 and was required for its activation. Furthermore, we demonstrated that overexpression of DOK3 and TBK1 could significantly enhance ifn-β promoter activity. DOK3 was also shown to bind TRAF3, and the binding of TRAF3 and TBK1 to DOK3 required the tyrosine-rich C-terminal domain of DOK3. We further revealed that DOK3 was phosphorylated by Bruton's tyrosine kinase. Hence, DOK3 plays a critical and positive role in TLR3 signaling by enabling TRAF3/TBK1 complex formation and facilitating TBK1 and IFN regulatory factor 3 activation and the induction of IFN-β production.

Selenium suppresses the activation of transcription factor NF-κB and IRF3 induced by TLR3 or TLR4 agonists

Toll-like receptors (TLRs) play an important role in recognition of microbial components and induce innate immune responses by recognizing invading microbial pathogens leading to the activation of the adaptive immune responses. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and TRIF-dependent pathways leading to the expression of pro-inflammatory cytokines and type I interferons (IFNs). The MyD88- and TRIF-dependent pathways lead to the activation of NF-κB and IRF3 through the activation of IKK-β and TBK1, respectively. Selenium is an essential trace element nutrient possessing anticarcinogenic properties. Here, we attempted to identify the molecular targets of selenium in TLR signaling pathways. Selenium inhibited NF-κB activation induced by poly[I:C] (TLR3 agonist), LPS (TLR4 agonist) or overexpression of MyD88 or IKK-β which is the key kinase of MyD88-dependent signaling pathway. Selenium inhibited IRF3 activation induced by poly[I:C], LPS or the overexpression of TRIF or TBK1. Selenium also suppressed the expression of COX-2 and iNOS and the endogenous IFNβ mRNA induced by poly[I:C] or LPS. Therefore, our results suggest that selenium can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs leading to decreased inflammatory gene expression.

NEMO: An Adaptive Regulator

STKE Nuclear factor κB (NF-κB), a transcription factor critical in the immune response to pathogens, is maintained in the inactive state by an inhibitory protein (IκB). The IκB kinase (IKK) complex activates NF-κB by dissociating IκB, and NEMO, also known as IKKγ, is a regulatory subunit of this complex. Zhao et al. show that in addition to this role in NF-κB activation, NEMO helps to activate the interferon regulatory factors IRF3 and IRF7, which, in combination with NF-κB and activator protein 1, stimulate the transcription of type I interferons (IFN-α and -β) in virally infected cells. Experiments with mouse embryo fibroblasts deficient in the gene encoding NEMO showed that the efficient production of IFN-α and the subsequent expression of signal transducer and activator of transcription 1 (STAT1) required NEMO, but not IKKβ (a subunit required for NF-κB activation), suggesting that NEMO was not acting through the NF-κB pathway. Reporter gene assays also demonstrated the dependence of IRF3- and IRF7-mediated gene expression on NEMO. In the NEMO knockout cells, overexpression of TBK1 (an IKK-related kinase that is upstream of IRF3 and IRF7 activation) restored reporter gene activation, and NEMO was also required for activation of the kinase activity of TBK1, placing NEMO upstream of TBK1 in the path to IRF3 and IRF7 activation. However, NEMO did not interact directly with TBK1 (or the related kinase IKKϵ); instead, immunoprecipitation studies with transfected cells showed that TANK (TRAF family member-associated NF-κB activator) was required for the formation of a trimeric complex of TANK, NEMO, and either IKKϵ or TBK1. Mutational and deletion analyses suggest that different regions of NEMO participate in NF-κB versus IRF activation. — NRG Nat. Immunol. 8 , 592 (2007).