Liquid phase separation of NEMO induced by polyubiquitin chains activates NF-κB.

The NF-κB essential modulator (NEMO) is a regulatory subunit of the IκB kinase (IKK) complex that phosphorylates the NF-κB inhibitors IκBs. NEMO mediates IKK activation by binding to polyubiquitin chains (polyUb). Here, we show that Lys63(K63)-linked or linear polyUb binding to NEMO robustly induced the formation of liquid-like droplets in which IKK was activated. This liquid phase separation of NEMO was driven by multivalent interactions between NEMO and polyUb. Both the NEMO ubiquitin-binding (NUB) domain and the zinc-finger (ZF) domain of NEMO mediated binding to polyUb and contributed to NEMO phase separation and IKK activation in cells. Moreover, NEMO mutations associated with human immunodeficiency impaired its phase separation. These results demonstrate that polyUb activates IKK and NF-κB signaling by promoting the phase separation of NEMO.

IκB kinase-α coordinates BRD4 and JAK/STAT signaling to subvert DNA damage-based anticancer therapy.

Activation of the IκB kinase (IKK) complex has recurrently been linked to colorectal cancer (CRC) initiation and progression. However, identification of downstream effectors other than NF-κB has remained elusive. Here, analysis of IKK-dependent substrates in CRC cells after UV treatment revealed that phosphorylation of BRD4 by IKK-α is required for its chromatin-binding at target genes upon DNA damage. Moreover, IKK-α induces the NF-κB-dependent transcription of the cytokine LIF, leading to STAT3 activation, association with BRD4 and recruitment to specific target genes. IKK-α abrogation results in defective BRD4 and STAT3 functions and consequently irreparable DNA damage and apoptotic cell death upon different stimuli. Simultaneous inhibition of BRAF-dependent IKK-α activity, BRD4, and the JAK/STAT pathway enhanced the therapeutic potential of 5-fluorouracil combined with irinotecan in CRC cells and is curative in a chemotherapy-resistant xenograft model. Finally, coordinated expression of LIF and IKK-α is a poor prognosis marker for CRC patients. Our data uncover a functional link between IKK-α, BRD4, and JAK/STAT signaling with clinical relevance.

IKKα Kinase Regulates the DNA Damage Response and Drives Chemo-resistance in Cancer.

Phosphorylated IKKα(p45) is a nuclear active form of the IKKα kinase that is induced by the MAP kinases BRAF and TAK1 and promotes tumor growth independent of canonical NF-κB signaling. Insights into the sources of IKKα(p45) activation and its downstream substrates in the nucleus remain to be defined. Here, we discover that IKKα(p45) is rapidly activated by DNA damage independent of ATM-ATR, but dependent on BRAF-TAK1-p38-MAPK, and is required for robust ATM activation and efficient DNA repair. Abolishing BRAF or IKKα activity attenuates ATM, Chk1, MDC1, Kap1, and 53BP1 phosphorylation, compromises 53BP1 and RIF1 co-recruitment to sites of DNA lesions, and inhibits 53BP1-dependent fusion of dysfunctional telomeres. Furthermore, IKKα or BRAF inhibition synergistically enhances the therapeutic potential of 5-FU and irinotecan to eradicate chemotherapy-resistant metastatic human tumors in vivo. Our results implicate BRAF and IKKα kinases in the DDR and reveal a combination strategy for cancer treatment.

TSG101 associates with PARP1 and is essential for PARylation and DNA damage-induced NF-κB activation.

In a genome-wide screening for components of the dsDNA-break-induced IKK-NF-κB pathway, we identified scores of regulators, including tumor susceptibility gene TSG101. TSG101 is essential for DNA damage-induced formation of cellular poly(ADP-ribose) (PAR). TSG101 binds to PARP1 and is required for PARP1 activation. This function of TSG101 is independent of its role in the ESCRT-I endosomal sorting complex. In the absence of TSG101, the PAR-dependent formation of a nuclear PARP1-IKKγ signalosome, which triggers IKK activation, is impaired. According to its requirement for PARP1 and NF-κB activation, TSG101-deficient cells are defective in DNA repair and apoptosis protection. Loss of TSG101 results in PARP1 trapping at damage sites and mimics the effect of pharmacological PARP inhibition. We also show that the loss of TSG101 in connection with inactivated tumor suppressors BRCA1/2 in breast cancer cells is lethal. Our results imply TSG101 as a therapeutic target to achieve synthetic lethality in cancer treatment.

The HTLV-I oncoprotein Tax inactivates the tumor suppressor FBXW7.

Human T-cell leukemia virus type 1 (HTLV-I) is the etiological agent of adult T-cell leukemia (ATL). Mutational analysis has demonstrated that the tumor suppressor, F-box and WD repeat domain containing 7 (FBXW7/FBW7/CDC4), is mutated in primary ATL patients. However, even in the absence of genetic mutations, FBXW7 substrates are stabilized in ATL cells, suggesting additional mechanisms can prevent FBXW7 functions. Here, we report that the viral oncoprotein Tax represses FBXW7 activity, resulting in the stabilization of activated Notch intracellular domain, c-MYC, Cyclin E, and myeloid cell leukemia sequence 1 (BCL2-related) (Mcl-1). Mechanistically, we demonstrate that Tax directly binds to FBXW7 in the nucleus, effectively outcompeting other targets for binding to FBXW7, resulting in decreased ubiquitination and degradation of FBXW7 substrates. In support of the nuclear role of Tax, a non-degradable form of the nuclear factor kappa B subunit 2 (NFκB2/p100) was found to delocalize Tax to the cytoplasm, thereby preventing Tax interactions with FBXW7 and Tax-mediated inhibition of FBXW7. Finally, we characterize a Tax mutant that is unable to interact with FBXW7, unable to block FBXW7 tumor suppressor functions, and unable to effectively transform fibroblasts. These results demonstrate that HTLV-I Tax can inhibit FBXW7 functions without genetic mutations to promote an oncogenic state. These results suggest that Tax-mediated inhibition of FBXW7 is likely critical during the early stages of the cellular transformation process. IMPORTANCE: F-box and WD repeat domain containing 7 (FBXW7), a critical tumor suppressor of human cancers, is frequently mutated or epigenetically suppressed. Loss of FBXW7 functions is associated with stabilization and increased expression of oncogenic factors such as Cyclin E, c-Myc, Mcl-1, mTOR, Jun, and Notch. In this study, we demonstrate that the human retrovirus human T-cell leukemia virus type 1 oncoprotein Tax directly interacts with FBXW7, effectively outcompeting other targets for binding to FBXW7, resulting in decreased ubiquitination and degradation of FBXW7 cellular substrates. We further demonstrate that a Tax mutant unable to interact with and inactivate FBXW7 loses its ability to transform primary fibroblasts. Collectively, our results describe a novel mechanism used by a human tumor virus to promote cellular transformation.

Bcl3 Phosphorylation by Akt, Erk2, and IKK Is Required for Its Transcriptional Activity.

Unlike prototypical IκB proteins, which are inhibitors of NF-κB RelA, cRel, and RelB dimers, the atypical IκB protein Bcl3 is primarily a transcriptional coregulator of p52 and p50 homodimers. Bcl3 exists as phospho-protein in many cancer cells. Unphosphorylated Bcl3 acts as a classical IκB-like inhibitor and removes p50 and p52 from bound DNA. Neither the phosphorylation site(s) nor the kinase(s) phosphorylating Bcl3 is known. Here we show that Akt, Erk2, and IKK1/2 phosphorylate Bcl3. Phosphorylation of Ser33 by Akt induces switching of K48 ubiquitination to K63 ubiquitination and thus promotes nuclear localization and stabilization of Bcl3. Phosphorylation by Erk2 and IKK1/2 of Ser114 and Ser446 converts Bcl3 into a transcriptional coregulator by facilitating its recruitment to DNA. Cells expressing the S114A/S446A mutant have cellular proliferation and migration defects. This work links Akt and MAPK pathways to NF-κB through Bcl3 and provides mechanistic insight into how Bcl3 functions as an oncoprotein through collaboration with IKK1/2, Akt, and Erk2.

IKKα-STAT3-S727 axis: a novel mechanism in DOX-induced cardiomyopathy.

Doxorubicin (DOX) is an effective chemotherapeutic drug, but its use can lead to cardiomyopathy, which is the leading cause of mortality among cancer patients. Macrophages play a role in DOX-induced cardiomyopathy (DCM), but the mechanisms undlerlying this relationship remain unclear. This study aimed to investigate how IKKα regulates macrophage activation and contributes to DCM in a mouse model. Specifically, the role of macrophage IKKα was evaluated in macrophage-specific IKKα knockout mice that received DOX injections. The findings revealed increased expression of IKKα in heart tissues after DOX administration. In mice lacking macrophage IKKα, myocardial injury, ventricular remodeling, inflammation, and proinflammatory macrophage activation worsened in response to DOX administration. Bone marrow transplant studies confirmed that IKKα deficiency exacerbated cardiac dysfunction. Macrophage IKKα knockout also led to mitochondrial damage and metabolic dysfunction in macrophages, thereby resulting in increased cardiomyocyte injury and oxidative stress. Single-cell sequencing analysis revealed that IKKα directly binds to STAT3, leading to the activation of STAT3 phosphorylation at S727. Interestingly, the inhibition of STAT3-S727 phosphorylation suppressed both DCM and cardiomyocyte injury. In conclusion, the IKKα-STAT3-S727 signaling pathway was found to play a crucial role in DOX-induced cardiomyopathy. Targeting this pathway could be a promising therapeutic strategy for treating DOX-related heart failure.

A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation.

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-κB pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between the N- and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational changes in NEMO.

The host antiviral protein SAMHD1 suppresses NF-κB activation by interacting with the IKK complex during inflammatory responses and viral infection.

Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) inhibits HIV-1 replication in nondividing cells by reducing the intracellular dNTP pool. SAMHD1 also suppresses NF-κB activation induced by inflammatory stimuli and viral infections. Specifically, SAMHD1-mediated reduction of NF-κB inhibitory protein (IκBα) phosphorylation is important for the suppression of NF-κB activation. However, while the inhibitors of NF-κB kinase subunit alpha and beta (IKKα and IKKß) regulate IκBα phosphorylation, the mechanism by which SAMHD1 regulates phosphorylation of IκBα remains unclear. Here, we report that SAMHD1 suppresses phosphorylation of IKKα/ß/γ via interaction with IKKα and IKKß, thus inhibiting subsequent phosphorylation of IκBα in monocytic THP-1 cells and differentiated nondividing THP-1 cells. We show that knockout of SAMHD1 enhanced phosphorylation of IKKα, IKKß, and IKKγ in THP-1 cells treated with the NF-κB activator lipopolysaccharide or infected with Sendai virus and SAMHD1 reconstitution inhibited phosphorylation of IKKα/ß/γ in Sendai virus-infected THP-1 cells. We demonstrate that endogenous SAMHD1 interacted with IKKα and IKKß in THP-1 cells and recombinant SAMHD1 bound to purified IKKα or IKKß directly in vitro. Mapping of these protein interactions showed that the HD domain of SAMHD1 interacts with both IKKα and IKKß and that the kinase domain of IKKα and the ubiquitin-like domain of IKKß are required for their interactions with SAMHD1, respectively. Moreover, we found that SAMHD1 disrupts the interaction between upstream kinase TAK1 and IKKα or IKKß. Our findings identify a new regulatory mechanism by which SAMHD1 inhibits phosphorylation of IκBα and NF-κB activation.

Stress-induced pseudokinase TRB3 augments IL1ß signaling by interacting with Flightless homolog 1.

Interleukin-1ß is one of the most potent inducers of beta cell inflammation in the lead-up to type 1 diabetes. We have previously reported that IL1ß-stimulated pancreatic islets from mice with genetic ablation of stress-induced pseudokinase TRB3(TRB3KO) show attenuated activation kinetics for the MAP3K MLK3 and JNK stress kinases. However, JNK signaling constitutes only a portion of the cytokine-induced inflammatory response. Here we report that TRB3KO islets also show a decrease in amplitude and duration of IL1ß-induced phosphorylation of TAK1 and IKK, kinases that drive the potent NF-κB proinflammatory signaling pathway. We observed that TRB3KO islets display decreased cytokine-induced beta cell death, preceded by a decrease in select downstream NF-κB targets, including iNOS/NOS2 (inducible nitric oxide synthase), a mediator of beta cell dysfunction and death. Thus, loss of TRB3 attenuates both pathways required for a cytokine-inducible, proapoptotic response in beta cells. In order to better understand the molecular basis of TRB3-enhanced, post-receptor IL1ß signaling, we interrogated the TRB3 interactome using coimmunoprecipitation followed by mass spectrometry to identify immunomodulatory protein Flightless homolog 1 (Fli1) as a novel, TRB3-interacting protein. We show that TRB3 binds and disrupts Fli1-dependent sequestration of MyD88, thereby increasing availability of this most proximal adaptor required for IL1ß receptor-dependent signaling. Fli1 sequesters MyD88 in a multiprotein complex resulting in a brake on the assembly of downstream signaling complexes. By interacting with Fli1, we propose that TRB3 lifts the brake on IL1ß signaling to augment the proinflammatory response in beta cells.

Regulation mechanisms of CARMA1-Bcl10-MALT1 complex assembly inferred from the analysis of TRAF6-deficient cells.

The CARMA1-Bcl10-MALT1 (CBM) signalosome is a crucial module of NF-κB activation in B cell receptor (BCR) signaling. Biophysical studies have shown that the E3 ubiquitin ligase TRAF6 cooperatively modifies the CBM signalosome; however, the specific details regarding how TRAF6 is involved in BCR signal-induced CBM formation remain unclear. In this study, we aimed to reveal the influences of TRAF6 on CBM formation and TAK1 and IKK activities using DT40 B cells which lack all the exons of TRAF6. In TRAF6-null cells we found: (i) attenuation of TAK1 activity and abolishment of IKK activity and (ii) sustained binding of CARMA1 to Bcl10. To account for the molecular mechanism causing these dynamics, we performed a mathematical model analysis. The mathematical model analysis showed that the regulation of IKK activation by TRAF6 can reproduce TAK1 and IKK activities in TRAF6 null cells, and that the TRAF6 related signal-dependent inhibitor suppresses CARMA1 binding to Bcl10 in wild-type cells. These results suggest that TRAF6 contributes to the positive regulation of IKK activation via TAK1, alongside the negative signal-dependent regulation of CARMA1 binding to Bcl10.

Mitigating sTNF/TNFR1 activation on VGluT2 + spinal cord interneurons improves immune function after mid-thoracic spinal cord injury.

Spinal cord injury (SCI) is a devastating condition with 250,000 to 500,000 new cases globally each year. Respiratory infections, e.g., pneumonia and influenza are the leading cause of death after SCI. Unfortunately, there is a poor understanding of how altered neuro-immune communication impacts an individual's outcome to infection. In humans and rodents, SCI leads to maladaptive changes in the spinal-sympathetic reflex (SSR) circuit which is crucial to sympathetic function. The cause of the impaired immune function may be related to harmful neuroinflammation which is detrimental to homeostatic neuronal function, aberrant plasticity, and hyperexcitable circuits. Soluble tumor necrosis factor (sTNF) is a pro-inflammatory cytokine that is elevated in the CNS after SCI and remains elevated for several months after injury. By pharmacologically attenuating sTNF in the CNS after SCI we were able to demonstrate improved immune function. Furthermore, when we investigated the specific cellular population which may be involved in altered neuro-immune communication we reported that excessive TNFR1 activity on excitatory INs promotes immune dysfunction. Furthermore, this observation is NF-kß dependent in VGluT2 + INs. Our data is the first report of a target within the CNS, TNFR1, that contributes to SCI-induced immune dysfunction after T9-SCI and is a potential avenue for future therapeutics.

Protein Kinases in Obesity, and the Kinase-Targeted Therapy.

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified as dual-specificity kinases and dual-specificity phosphatases. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases and play an important role in obesity. Impairment of insulin signaling in obesity is largely mediated by the activation of the inhibitor of kappa B-kinase beta and the c-Jun N-terminal kinase (JNK). Oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular levels. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. To alleviate lipotoxicity and insulin resistance, promising targets are pharmacologically inhibited. Nifedipine, calcium channel blocker, stimulates lipogenesis and adipogenesis by downregulating AMPK and upregulating mTOR, which thereby enhances lipid storage. Contrary to the nifedipine, metformin activates AMPK, increases fatty acid oxidation, suppresses fatty acid synthesis and deposition, and thus alleviates lipotoxicity. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2 alpha kinase (PERK), and activating transcription factor-6. The transcriptional regulation of adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. An increase in intracellular oxidative stress can promote PKC-ß activation. Activated PKC-ß induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhance triglyceride accumulation and lipotoxicity. Liraglutide attenuates mitochondrial dysfunction and reactive oxygen species generation. Co-treatment of antiobesity and antidiabetic herbal compound, berberine with antipsychotic drug olanzapine decreases the accumulation of triglyceride. While low-dose rapamycin, metformin, amlexanox, thiazolidinediones, and saroglitazar protect against insulin resistance, glucagon-like peptide-1 analog liraglutide inhibits palmitate-induced inflammation by suppressing mTOR complex 1 (mTORC1) activity and protects against lipotoxicity.

Design and Synthesis of Novel Aminoindazole-pyrrolo[2,3-b]pyridine Inhibitors of IKKα That Selectively Perturb Cellular Non-Canonical NF-κB Signalling.

The inhibitory-kappaB kinases (IKKs) IKKα and IKKß play central roles in regulating the non-canonical and canonical NF-κB signalling pathways. Whilst the proteins that transduce the signals of each pathway have been extensively characterised, the clear dissection of the functional roles of IKKα-mediated non-canonical NF-κB signalling versus IKKß-driven canonical signalling remains to be fully elucidated. Progress has relied upon complementary molecular and pharmacological tools; however, the lack of highly potent and selective IKKα inhibitors has limited advances. Herein, we report the development of an aminoindazole-pyrrolo[2,3-b]pyridine scaffold into a novel series of IKKα inhibitors. We demonstrate high potency and selectivity against IKKα over IKKß in vitro and explain the structure-activity relationships using structure-based molecular modelling. We show selective target engagement with IKKα in the non-canonical NF-κB pathway for both U2OS osteosarcoma and PC-3M prostate cancer cells by employing isoform-related pharmacodynamic markers from both pathways. Two compounds (SU1261 [IKKα Ki = 10 nM; IKKß Ki = 680 nM] and SU1349 [IKKα Ki = 16 nM; IKKß Ki = 3352 nM]) represent the first selective and potent pharmacological tools that can be used to interrogate the different signalling functions of IKKα and IKKß in cells. Our understanding of the regulatory role of IKKα in various inflammatory-based conditions will be advanced using these pharmacological agents.

Connecting GSK-3ß Inhibitory Activity with IKK-ß or ROCK-1 Inhibition to Target Tau Aggregation and Neuroinflammation in Alzheimer's Disease-Discovery, In Vitro and In Cellulo Activity of Thiazole-Based Inhibitors.

GSK-3ß, IKK-ß, and ROCK-1 kinases are implicated in the pathomechanism of Alzheimer's disease due to their involvement in the misfolding and accumulation of amyloid ß (Aß) and tau proteins, as well as inflammatory processes. Among these kinases, GSK-3ß plays the most crucial role. In this study, we present compound 62, a novel, remarkably potent, competitive GSK-3ß inhibitor (IC50 = 8 nM, Ki = 2 nM) that also exhibits additional ROCK-1 inhibitory activity (IC50 = 2.3 µM) and demonstrates anti-inflammatory and neuroprotective properties. Compound 62 effectively suppresses the production of nitric oxide (NO) and pro-inflammatory cytokines in the lipopolysaccharide-induced model of inflammation in the microglial BV-2 cell line. Furthermore, it shows neuroprotective effects in an okadaic-acid-induced tau hyperphosphorylation cell model of neurodegeneration. The compound also demonstrates the potential for further development, characterized by its chemical and metabolic stability in mouse microsomes and fair solubility.

Mechanistic insights into the activation of the IKK kinase complex by the Kaposi's sarcoma herpes virus oncoprotein vFLIP.

Constitutive activation of the canonical NF-κB signaling pathway is a major factor in Kaposi's sarcoma-associated herpes virus pathogenesis where it is essential for the survival of primary effusion lymphoma. Central to this process is persistent upregulation of the inhibitor of κB kinase (IKK) complex by the virally encoded oncoprotein vFLIP. Although the physical interaction between vFLIP and the IKK kinase regulatory component essential for persistent activation, IKKγ, has been well characterized, it remains unclear how the kinase subunits are rendered active mechanistically. Using a combination of cell-based assays, biophysical techniques, and structural biology, we demonstrate here that vFLIP alone is sufficient to activate the IKK kinase complex. Furthermore, we identify weakly stabilized, high molecular weight vFLIP-IKKγ assemblies that are key to the activation process. Taken together, our results are the first to reveal that vFLIP-induced NF-κB activation pivots on the formation of structurally specific vFLIP-IKKγ multimers which have an important role in rendering the kinase subunits active through a process of autophosphorylation. This mechanism of NF-κB activation is in contrast to those utilized by endogenous cytokines and cellular FLIP homologues.

IKKα aggravates renal fibrogenesis by positively regulating the Wnt/ß-catenin pathway.

AKI (acute kidney injury) with maladaptive repair plays exacerbated role in renal fibrosis characterized by tubulointerstitial fibrosis. Previously, we reported that IKKα contributed to kidney regeneration and inhibited inflammation. Here, we first identified the role and mechanism of IKKα on TGF-ß1-induced fibrosis in human tubular epithelial cells and fibrotic kidneys. IKKα was up-regulated in kidney tubular epithelium in unilateral ureteral obstruction (UUO) and unilateral ischemic reperfusion injury (UIRI) mice. Immunohistochemical staining showed that IKKα was positively correlated with the extent of kidney fibrosis in tissue biopsies from chronic kidney disease (CKD) patients. Compared with wild-type controls, Ksp-IKKα-/- mice exhibited inactivated Wnt/ß-catenin pathway, decreased serum creatinine and interstitial fibrosis in the kidney after IRI. In TGF-ß1-stimulated human tubular epithelial cells, IKKα overexpression enhanced ß-catenin nuclear translocation. Blocking IKKα by siRNA specifically suppressed ß-catenin activation and downstream profibrotic genes such as fibronectin and α-smooth muscle actin (α-SMA). Taken together, our study demonstrated that IKKα aggravated renal fibrogenesis by activating Wnt/ß-catenin signalling pathway, providing a new target for the treatment of kidney fibrosis.

IKKα-Mediated Noncanonical NF-κB Signaling Is Required To Support Murine Gammaherpesvirus 68 Latency In Vivo.

Noncanonical NF-κB signaling is activated in B cells via the tumor necrosis factor (TNF) receptor superfamily members CD40, lymphotoxin ß receptor (LTßR), and B-cell-activating factor receptor (BAFF-R). The noncanonical pathway is required at multiple stages of B cell maturation and differentiation, including the germinal center reaction. However, the role of this pathway in gammaherpesvirus latency is not well understood. Murine gammaherpesvirus 68 (MHV68) is a genetically tractable system used to define pathogenic determinants. Mice lacking the BAFF-R exhibit defects in splenic follicle formation and are greatly reduced for MHV68 latency. We report a novel approach to disrupt noncanonical NF-κB signaling exclusively in cells infected with MHV68. We engineered a recombinant virus that expresses a dominant negative form of IκB kinase α (IKKα), named IKKα-SA, with S176A and S180A mutations that prevent phosphorylation by NF-κB-inducing kinase (NIK). We controlled for the transgene insertion by introducing two all-frame stop codons into the IKKα-SA gene. The IKKα-SA mutant but not the IKKα-SA.STOP control virus impaired LTßR-mediated activation of NF-κB p52 upon fibroblast infection. IKKα-SA expression did not impact replication in primary fibroblasts or in the lungs of mice following intranasal inoculation. However, the IKKα-SA mutant was severely defective in the colonization of the spleen and in the establishment of latency compared to the IKKα-SA.STOP control and wild-type (WT) MHV68 at 16 days postinfection (dpi). Reactivation was undetectable in splenocytes infected with the IKKα-SA mutant, but reactivation in peritoneal cells was not impacted by IKKα-SA. Taken together, the noncanonical NF-κB signaling pathway is essential for the establishment of latency in the secondary lymphoid organs of mice infected with the murine gammaherpesvirus pathogen MHV68. IMPORTANCE The latency programs of the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) are associated with B cell lymphomas. It is critical to understand the signaling pathways that are used by gammaherpesviruses to establish and maintain latency in primary B cells. We used a novel approach to block noncanonical NF-κB signaling only in the infected cells of mice. We generated a recombinant virus that expresses a dominant negative mutant of IKKα that is nonresponsive to upstream activation. Latency was reduced in a route- and cell type-dependent manner in mice infected with this recombinant virus. These findings identify a significant role for the noncanonical NF-κB signaling pathway that might provide a novel target to prevent latent infection of B cells with oncogenic gammaherpesviruses.

Genome-wide identification and functional characterization of inhibitor of nuclear factor-κB (IκB) kinase (IKK) in turbot (Scophthalmus maximus).

The inhibitor of nuclear factor-κB (IκB) kinase (IKK) is involved in a variety of intracellular cell signaling pathways and is an important component of the NF-κB signaling pathway. IKK genes have been suggested to play important roles in the innate immune response to pathogen infection in both vertebrates and invertebrates. However, little information is available about IKK genes in turbot (Scophthalmus maximus). In this study, six IKK genes were identified including SmIKKα, SmIKKα2, SmIKKß, SmIKKε, SmIKKγ, and SmTBK1. The IKK genes of turbot showed the highest identity and similarity with Cynoglossus semilaevis. Then, phylogenetic analysis showed that the IKK genes of turbot were most closely related to C. semilaevis. In addition, IKK genes were widely expressed in all the examined tissues. Meanwhile, the expression patterns of IKK genes were investigated by QRT-PCR after Vibrio anguillarum and Aeromonas salmonicida infection. The results showed that IKK genes had varying expression patterns in mucosal tissues after bacteria infection, indicating that they may play key roles in maintaining the integrity of the mucosal barrier. Subsequently, protein and protein interaction (PPI) network analysis showed that most proteins interacting with IKK genes were located in the NF-κB signaling pathway. Finally, the double luciferase report and overexpression experiments showed that SmIKKα/SmIKKα2/SmIKKß involved in the activation of NF-κB in turbot. In summary, our results suggested that IKK genes of turbot played important roles in the innate immune response of teleost, and provide valuable information for further study of the function of IKK genes.

Costunolide alleviates atherosclerosis in high-fat diet-fed ApoE-/- mice through covalently binding to IKKß and inhibiting NF-κB-mediated inflammation.

Costunolide (CTD) is a sesquiterpene lactone isolated from costus root and exhibits various biological activities including anti-inflammation. Since atherosclerosis is a chronic inflammatory disease, we herein investigated the anti-atherosclerotic effects of CTD and the underlying mechanism. Atherosclerosis was induced in ApoE-/- mice by feeding them with a high-fat diet (HFD) for 8 weeks, followed by administration of CTD (10, 20 mg ·kg-1·d-1, i.g.) for 8 weeks. We showed that CTD administration dose-dependently alleviated atherosclerosis in HFD-fed ApoE-/- mice. Furthermore, we found that CTD dose-dependently reduced inflammatory responses in aortas of the mice, as CTD prevented infiltration of inflammatory cells in aortas and attenuated oxLDL uptake in macrophages, leading to reduced expression of pro-inflammatory and pro-fibrotic molecules in aortas. Similar results were observed in oxLDL-stimulated mouse primary peritoneal macrophages (MPMs) in vitro. We showed that pretreatment with CTD (2.5, 5. 10 µM) restrained oxLDL-induced inflammatory responses in MPMs by blocking pro-inflammatory NF-κB/p65 signaling pathway. We further demonstrated that CTD inactivated NF-κB via covalent binding to cysteine 179 on IKKß, a canonical upstream regulator of NF-κB, reducing its phosphorylation and leading to conformational change in the active loop of IKKß. Our results discover IKKß as the target of CTD for its anti-inflammatory activity and elucidate a molecular mechanism underlying the anti-atherosclerosis effect of CTD. CTD is a potentially therapeutic candidate for retarding inflammatory atherosclerotic diseases.