Cucurbitacin B suppresses the transactivation activity of RelA/p65

Cucurbitacin B, a natural triterpenoid is well-known for its strong anticancer activity, and recent studies showed that the compound inhibits JAK/STAT3 pathway. In this study, we demonstrate for the first time that cucurbitacin B is also a potent inhibitor of NF-κB activation. Our results showed that cucurbitacin B inhibited TNF-α-induced expression of NF-κB reporter gene and NF-κB target genes in a dose-dependent manner, however, it did not prevent either stimuli-induced degradation of IκBα or nuclear translocation and DNA-binding activity of NF-κB. On the other hand, cucurbitacin B dose-dependently suppressed not only NF-κB activation induced by overexpression of RelA/p65 but also transactivation activity of RelA/p65 subunit of NF-κB. Consistently, treatment of HeLa cells with the compound significantly suppressed TNF-α-induced activation of Akt and phosphorylation of Ser536 in RelA/p65, which is required for transactivation activity. Consequently, cucurbitacin B inhibited TNF-α-induced expression of NF-κB-dependent anti-apoptotic proteins such as c-IAP1, c-IAP2, XIAP, TRAF1, and TRAF2 and sensitized TNF-α-induced cell death. Taken together, our results demonstrated that cucurbitacin B could be served as a valuable candidate for the intervention of NF-κB-dependent pathological condition such as cancer.     

Antioxidant c-FLIP inhibits Fas ligand-induced NF-kappaB activation in a phosphatidylinositol 3-kinase/Akt-dependent manner

Fas ligand (FasL) belongs to the TNF family of death ligands, and its binding to the FasR leads to activation of several downstream signaling pathways and proteins, including NF-κB and PI3K/Akt. However, it is not known whether cross-talk exists between NF-κB and PI3K/Akt in the context of FasL signaling. We demonstrate using both human renal epithelial 293T cells and Jurkat T-lymphocyte cells that although FasL activates both Akt and NF-κB, Akt inhibits FasL-dependent NF-κB activity in a reactive oxygen species-dependent manner. Cellular FLICE-inhibitory protein (c-FLIP), an antioxidant and an important component of the death-inducing signaling complex, also represses NF-κB upstream of the regulatory IκB kinase-γ protein subunit in the NF-κB signaling pathway, and positive cross-talk exists between Akt and c-FLIP in the context of inhibition of FasL-induced NF-κB activity. The presence of two death effector domains of c-FLIP and S-nitrosylation of its caspase-like domain were found to be important for mediating c-FLIP-dependent downregulation of NF-κB activity. Taken together, our study reveals a novel link between NF-κB and PI3K/Akt and establishes c-FLIP as an important regulator of FasL-mediated cell death.     

TGF-beta coordinately activates TAK1/MEK/AKT/NFkB and SMAD pathways to promote osteoclast survival

To better understand the roles of TGF-β in bone metabolism, we investigated osteoclast survival in response TGF-β and found that TGF-β inhibited apoptosis. We examined the receptors involved in promotion of osteoclast survival and found that the canonical TGF-β receptor complex is involved in the survival response. The upstream MEK kinase TAK1 was rapidly activated following TGF-β treatment. Since osteoclast survival involves MEK, AKT, and NFκB activation, we examined TGF-β effects on activation of these pathways and observed rapid phosphorylation of MEK, AKT, IKK, IκB, and NFκB. The timing of activation coincided with SMAD activation and dominant negative SMAD expression did not inhibit NFκB activation, indicating that kinase pathway activation is independent of SMAD signaling. Inhibition of TAK1, MEK, AKT, NIK, IKK, or NFκB repressed TGF-β-mediated osteoclast survival. Adenoviral-mediated TAK1 or MEK inhibition eliminated TGF-β-mediated kinase pathway activation and constitutively active AKT expression overcame apoptosis induction following MEK inhibition. TAK1/MEK activation induces pro-survival BclX_L expression and TAK1/MEK and SMAD pathway activation induces pro-survival Mcl-1 expression. These data show that TGF-β-induced NFκB activation is through TAK1/MEK-mediated AKT activation, which is essential for TGF-β to support of osteoclast survival.     

Distal NF-kB binding motif functions as an enhancer for nontypeable H. influenzae-induced DEFB4 regulation in epithelial cells

Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to proinflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5′-flanking region of DEFB4 contains several NF-κB binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-κB binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-κB activation mediated by IκKα/β−IκBα signaling. Deletion of the distal NF-κB binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-κB binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-κB binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-κB binds to the distal NF-κB binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-κB to the distal NF-κB binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells.     

Induction of apoptosis through extrinsic/intrinsic pathways and suppression of ERK/NF-κB signalling participate in anti-glioblastoma of imipramine

Glioblastomas are the most aggressive type of brain tumour, with poor prognosis even after standard treatment such as surgical resection, temozolomide and radiation therapy. The overexpression of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in glioblastomas is recognized as an important treatment target. Thus, an urgent need regarding glioblastomas is the development of a new, suitable agent that may show potential for the inhibition of extracellular signal-regulated kinase (ERK)/NF-κB–mediated glioblastoma progression. Imipramine, a tricyclic antidepressant, has anti-inflammatory actions against inflamed glial cells; additionally, imipramine can induce glioblastoma toxicity via the activation of autophagy. However, whether imipramine can suppress glioblastoma progression via the induction of apoptosis and blockage of ERK/NF-κB signalling remains unclear. The main purpose of this study was to investigate the effects of imipramine on apoptotic signalling and ERK/NF-κB–mediated glioblastoma progression by using cell proliferation (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT] assay), flow cytometry, Western blotting, and cell invasion/migration assay analysis in vitro. The ERK and NF-κB inhibitory capacity of imipramine is detected by NF-κB reporter gene assay and Western blotting. Additionally, a glioblastoma-bearing animal model was used to validate the therapeutic efficacy and general toxicity of imipramine. Our results demonstrated that imipramine successfully triggered apoptosis through extrinsic/intrinsic pathways and suppressed the invasion/migration ability of glioblastoma cells. Furthermore, imipramine effectively suppressed glioblastoma progression in vivo via the inhibition of the ERK/NF-κB pathway. In summary, imipramine is a potential anti-glioblastoma drug which induces apoptosis and has the capacity to inhibit ERK/NF-κB signalling.     

p97/VCP promotes Cullin‐RING‐ubiquitin‐ligase/proteasome‐dependent degradation of IκBα and the preceding liberation of RelA from ubiquitinated IκBα

Cullin‐RING‐ubiquitin‐ligase (CRL)‐dependent ubiquitination of the nuclear factor kappa B (NF‐κB) inhibitor IκBα and its subsequent degradation by the proteasome usually precede NF‐κB/RelA nuclear activity. Through removal of the CRL‐activating modification of their cullin subunit with the ubiquitin (Ub)‐like modifier NEDD8, the COP9 signalosome (CSN) opposes CRL Ub‐ligase activity. While RelA phosphorylation was observed to mediate NF‐κB activation independent of Ub‐proteasome‐pathway (UPP)‐dependent turnover of IκBα in some studies, a strict requirement of the p97/VCP ATPase for both, IκBα degradation and NF‐κB activation, was reported in others. In this study, we thus aimed to reconcile the mechanism for tumour necrosis factor (TNF)‐induced NF‐κB activation. We found that inducible phosphorylation of RelA is accomplished in an IKK‐complex‐dependent manner within the NF‐κB/RelA‐IκBα‐complex contemporaneous with the phosphorylation of IκBα, and that RelA phosphorylation is not sufficient to dissociate NF‐κB/RelA from IκBα. Subsequent to CRL‐dependent IκBα ubiquitination functional p97/VCP is essentially required for efficient liberation of (phosphorylated) RelA from IκBα, preceding p97/VCP‐promoted timely and efficient degradation of IκBα as well as simultaneous NF‐κB/RelA nuclear translocation. Collectively, our data add new facets to the knowledge about maintenance of IκBα and RelA expression, likely depending on p97/VCP‐supported scheduled basal NF‐κB activity, and the mechanism of TNF‐induced NF‐κB activation.     

Endothelin-1 promotes MMP-13 production and migration in human chondrosarcoma cells through FAK/PI3K/Akt/mTOR pathways

Tumor malignancy is associated with several cellular properties including proliferation and ability to metastasize. Endothelin-1 (ET-1) the most potent vasoconstrictor plays a crucial role in migration and metastasis of human cancer cells. We found that treatment of human chondrosarcoma (JJ012 cells) with ET-1 increased migration and expression of matrix metalloproteinase (MMP)-13. ET-1-mediated cell migration and MMP-13 expression were reduced by pretreatment with inhibitors of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR), as well as the NF-κB inhibitor and the IκB protease inhibitor. In addition, ET-1 treatment induced phosphorylation of FAK, PI3K, AKT, and mTOR, and resulted in increased NF-κB-luciferase activity that was inhibited by a specific inhibitor of PI3K, Akt, mTOR, and NF-κB cascades. Taken together, these results suggest that ET-1 activated FAK/PI3K/AKT/mTOR, which in turn activated IKKα/β and NF-κB, resulting in increased MMP-13 expression and migration in human chondrosarcoma cells.     

Thromboxane A2 increases endothelial permeability through upregulation of interleukin-8

Thromboxane A₂ (TXA₂), a major prostanoid formed from prostaglandin H₂ by thromboxane synthase, is involved in the pathogenesis of a variety of vascular diseases. In this study, we report that TXA₂ mimetic U46619 significantly increases the endothelial permeability both in vitro and in vivo. U46619 enhanced the expression and secretion of interleukin-8 (IL-8), a major inducer of vascular permeability, in endothelial cells. Promoter analysis showed that the U46619-induced expression of IL-8 was mainly regulated by nuclear factor-κB (NF-κB). U46619 induced the activation of NF-κB through IκB kinase (IKK) activation, IκB phosphorylation and NF-κB nuclear translocation. Furthermore, the inhibition of IL-8 or blockade of the IL-8 receptor attenuated the U46619-induced endothelial cell permeability by modulating the cell-cell junctions. Overall, these results suggest that U46619 promotes vascular permeability through the production of IL-8 via NF-κB activation in endothelial cells.     

NF-kappaB protects human papillomavirus type 38 E6/E7-immortalized human keratinocytes against tumor necrosis factor alpha and UV-mediated apoptosis

Constitutive activation of NF-κB signaling is a key event in virus- and non-virus-induced carcinogenesis. We have previously reported that cutaneous human papillomavirus type 38 (HPV38) displays transforming properties in in vitro and in vivo experimental models. However, the involvement of NF-κB signaling in HPV38-induced cell growth transformation remains to be determined. In this study, we showed that HPV38 E6 and E7 activate NF-κB and that inhibition of the pathway with the IκBα superrepressor sensitizes HPV38E6E7-immortalized human keratinocytes to tumor necrosis factor alpha (TNF-α)- and UVB radiation-mediated apoptosis. Accordingly, inhibition of NF-κB signaling resulted in the downregulation of NF-κB-regulated antiapoptotic genes, including cIAP1, cIAP2, and xIAP genes. These findings demonstrate a critical role of NF-κB activity in the survival of HPV38E6E7-immortalized human keratinocytes exposed to cytokine or UV radiation. Our data provide additional evidence for cooperation between beta HPV infection and UV irradiation in skin carcinogenesis.     

Regulation of adult mammalian intrinsic axonal regeneration by NF-κB/STAT3 signaling cascade

The inflammatory response is a critical regulator for the regeneration of axon following nervous system injury. Nuclear factor-kappa B (NF-κB) is characteristically known for its ubiquitous role in the inflammatory response. However, its functional role in adult mammalian axon growth remains elusive. Here, we found that the NF-κB signaling pathway is activated in adult sensory neurons through peripheral axotomy. Furthermore, inhibition of NF-κB in peripheral sensory neurons attenuated their axon growth in vitro and in vivo. Our results also showed that NF-κB modulated axon growth by repressing the phosphorylation of STAT3. Furthermore, activation of STAT3 significantly promoted adult optic nerve regeneration. Taken together, the findings of our study indicated that NF-κB/STAT3 cascade is a critical regulator of intrinsic axon growth capability in the adult nervous system.     

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF’s role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-κB and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-κB activity and phosphorylation of the inhibitor of kappa B (IκBα) increased in ischemic brains, but IRF3, inhibitor of κB kinase complex-ε (IKKε), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-κB activity or p-IκBα induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-κB signaling and brain injury after acute cerebral I/R.     

Saikosaponin-D induces the pyroptosis of lung cancer by increasing ROS and activating the NF-κB/NLRP3/caspase-1/GSDMD pathway

Saikosaponin-D (SSD), an active ingredient in Bupleurum chinense, exerts anticancer effects in various cancers by inhibiting cancer proliferation and inducing apoptosis. However, whether SSD can induce other forms of cell death is unknown. The current study aims to demonstrate that SSD can induce pyroptosis in non-small-cell lung cancer. In this study, HCC827 and A549 non-small-cell lung cancer cells were treated with different concentrations of SSD for 1.5 h. HE and TUNEL staining were used to verify cell damage caused by SSD. Immunofluorescence and western blotting were performed to verify the effect of SSD on the NF-κB/NLRP3/caspase-1/gasdermin D (GSDMD) pathway. Changes in inflammatory factors were detected by ELISAs. Finally, the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) was introduced to verify that SSD induces pyroptosis through the ROS/NF-κB pathway. The results of the HE and TUNEL staining showed that SSD resulted in balloon-like swelling of NSCLC cells accompanied by increased DNA damage. Immunofluorescence and western blot assays confirmed that SSD treatment activated the NLRP3/caspase-1/GSDMD pathway, stimulated an increase in ROS levels and activated NF-κB in lung cancer cells. The ROS scavenger N-acetylcysteine significantly attenuated SSD-induced NF-κB/NLRP3/caspase-1/GSDMD pathway activation and inhibited the release of the inflammatory cytokines IL-1β and IL-18. In conclusion, SSD induced lung cancer cell pyroptosis by inducing ROS accumulation and activating the NF-κB/NLRP3/caspase-1/GSDMD pathway. These experiments lay the foundation for the application of SSD in the treatment of non-small-cell lung cancer and regulation of the lung cancer immune microenvironment.     

NDR1/STK38 potentiates NF‐κB activation by its kinase activity

Human NDR1/STK38 belongs to the nuclear‐Dbf2‐related (NDR) family of Ser/Thr kinases. It has been implicated to function in centrosome duplication, control of cell cycle and apoptosis. However, the mechanism of NDR1 signaling pathway remains largely elusive. Here, we report a novel role of NDR1 in NF‐κB activation. By overexpression, NDR1 potentiates NF‐κB activation induced by TNFα, whereas knockdown of NDR1 expression inhibits NF‐κB activation induced by TNFα. Coimmunoprecipitation shows that NDR1 interacts with multiple signal components except p65 in NF‐κB signaling pathway. Furthermore, both phosphorylation and kinase dead mutants of NDR1 lose their synergistic effects on TNFα‐induced NF‐κB activation. siRNA oligo against NDR1 and kinase dead mutant as well mainly block the NF‐κB activation induced by TRAF2 but not RIP1. Furthermore, kinase dead mutant of NDR1 fails to interact with TRAF2. Taken together, our findings suggest an unknown function of NDR1, which may regulate NF‐κB activation by its kinase activity. Copyright © 2012 John Wiley & Sons, Ltd.