VRK2 activates TNFα/NF-κB signaling by phosphorylating IKKβ in pancreatic cancer

NF-κB signaling is active in more than 50% of patients with pancreatic cancer and plays an important role in promoting the progression of pancreatic cancer. Revealing the activation mechanism of NF-κB signaling is important for the treatment of pancreatic cancer. In this study, the regulation of TNFα/NF-κB signaling by VRK2 (vaccinia-related kinase 2) was investigated. The levels of VRK2 protein were examined by immunohistochemistry (IHC). The functions of VRK2 in the progression of pancreatic cancer were examined using CCK8 assay, anchorage-independent assay, EdU assay and tumorigenesis assay. The regulation of VRK2 on the NF-κB signaling was investigated by immunoprecipitation and invitro kinase assay. It was discovered in this study that the expression of VRK2 was upregulated in pancreatic cancer and that the VRK2 expression level was significantly correlated with the pathological characteristics and the survival time of patients. VRK2 promoted the growth, sphere formation and subcutaneous tumorigenesis of pancreatic carcinoma cells as well as the organoid growth derived from the pancreatic cancer mouse model. Investigation of the molecular mechanism indicated that VRK2 interacts with IKKβ, phosphorylating its Ser177 and Ser181 residues and thus activating the TNFα/NF-κB signaling pathway. An IKKβ inhibitors abolished the promotive effect of VRK2 on the growth of organoids. The findings of this study indicate that VRK2 promotes the progression of pancreatic cancer by activating the TNFα/NF-κB signaling pathway, suggesting that VRK2 is a potential therapeutic target for pancreatic cancer.     

TRIM22 activates NF-κB signaling in glioblastoma by accelerating the degradation of IκBα

NF-κB signaling plays a critical role in tumor growth and treatment resistance in GBM as in many other cancers. However, the molecular mechanisms underlying high, constitutive NF-κB activity in GBM remains to be elucidated. Here, we screened a panel of tripartite motif (TRIM) family proteins and identified TRIM22 as a potential activator of NF-κB using an NF-κB driven luciferase reporter construct in GBM cell lines. Knockout of TRIM22 using Cas9-sgRNAs led to reduced GBM cell proliferation, while TRIM22 overexpression enhanced proliferation of cell populations, in vitro and in an orthotopic xenograft model. However, two TRIM22 mutants, one with a critical RING-finger domain deletion and the other with amino acid changes at two active sites of RING E3 ligase (C15/18A), were both unable to promote GBM cell proliferation over controls, thus implicating E3 ligase activity in the growth-promoting properties of TRIM22. Co-immunoprecipitations demonstrated that TRIM22 bound a negative regulator of NF-κB, NF-κB inhibitor alpha (IκBα), and accelerated its degradation by inducing K48-linked ubiquitination. TRIM22 also formed a complex with the NF-κB upstream regulator IKKγ and promoted K63-linked ubiquitination, which led to the phosphorylation of both IKKα/β and IκBα. Expression of a non-phosphorylation mutant, srIκBα, inhibited the growth-promoting properties of TRIM22 in GBM cell lines. Finally, TRIM22 was increased in a cohort of primary GBM samples on a tissue microarray, and high expression of TRIM22 correlated with other clinical parameters associated with progressive gliomas, such as wild-type IDH1 status. In summary, our study revealed that TRIM22 activated NF-κB signaling through posttranslational modification of two critical regulators of NF-κB signaling in GBM cells.Subject terms: CNS cancer, Oncogenes, Ubiquitin ligases     

Zoledronic acid modulates osteoclast apoptosis through activation of the NF-κB signaling pathway in ovariectomized rats

Bone mass loss (osteoporosis) seen in postmenopausal women is an adverse factor for implant denture. Using an ovariectomized rat model, we studied the mechanism of estrogen-deficiency-caused bone loss and the therapeutic effect of Zoledronic acid. We observed that ovariectomized-caused resorption of bone tissue in the mandible was evident at four weeks and had not fully recovered by 12 weeks post-ovariectomized compared with the sham-operated controls. Further evaluation with a TUNEL assay showed ovariectomized enhanced apoptosis of osteoblasts but inhibited apoptosis of osteoclasts in the mandible. Zoledronic acid given subcutaneously as a single low dose was shown to counteract both of these ovariectomized effects. Immunohistochemical staining showed that ovariectomized induced the protein levels of RANKL and the 65-kD subunit of the NF-κB complex mainly in osteoclasts, as confirmed by staining for TRAP, a marker for osteoclasts, whereas zoledronic acid inhibited these inductions. Western blotting showed that the levels of RANKL, p65, as well as the phosphorylated form of p65, and IκB-α were all higher in the ovariectomized group than in the sham and ovariectomized + zoledronic acid groups at both the 4th- and 12th-week time points in the mandible. These data collectively suggest that ovariectomized causes bone mass loss by enhancing apoptosis of osteoblasts and inhibiting apoptosis of osteoclasts. In osteoclasts, these cellular effects may be achieved by activating RANKL-NF-κB signalling. Moreover, zoledronic acid elicits its therapeutic effects in the mandible by counteracting these cellular and molecular consequences of ovariectomized.     

DNA-Dependent Protein Kinase Phosphorylation of IκBα and IκBβ Regulates NF-κB DNA Binding Properties

Regulation of the IκBα and IκBβ proteins is critical for modulating NF-κB-directed gene expression. Both IκBα and IκBβ are substrates for cellular kinases that phosphorylate the amino and carboxy termini of these proteins and regulate their function. In this study, we utilized a biochemical fractionation scheme to purify a kinase activity which phosphorylates residues in the amino and carboxy termini of both IκBα and IκBβ. Peptide microsequence analysis by capillary high-performance liquid chromatography ion trap mass spectroscopy revealed that this kinase was the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK phosphorylates serine residue 36 but not serine residue 32 in the amino terminus of IκBα and also phosphorylates threonine residue 273 in the carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of IκBα, murine severe combined immunodeficiency (SCID) cell lines which lack the DNA-PKcs gene were analyzed. Gel retardation analysis using extract prepared from these cells demonstrated constitutive nuclear NF-κB DNA binding activity, which was not detected in extracts prepared from SCID cells complemented with the human DNA-PKcs gene. Furthermore, IκBα that was phosphorylated by DNA-PK was a more potent inhibitor of NF-κB binding than nonphosphorylated IκBα. These results suggest that DNA-PK phosphorylation of IκBα increases its interaction with NF-κB to reduce NF-κB DNA binding properties.     

STING promotes senescence,apoptosis, and extracellular matrix degradation in osteoarthritis via the NF-κB signaling pathway

Damaged deoxyribonucleic acid (DNA) is a primary pathologic factor for osteoarthritis (OA); however, the mechanism by which DNA damage drives OA is unclear. Previous research demonstrated that the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) participates in DNA damage response. As a result, the current study aimed at exploring the role STING, which is the major effector in the cGAS-STING signaling casacde, in OA progress in vitro, as well as in vivo. In this study, the expression of STING was evaluated in the human and mouse OA tissues, and in chondrocytes exposed to interleukin-1 beta (IL-1β). The influences of STING on the metabolism of the extracellular matrix (ECM), apoptosis, and senescence, were assessed in STING overexpressing and knocking-down chondrocytes. Moreover, the NF-κB-signaling casacde and its role in the regulatory effects of STING on ECM metabolism, apoptosis, and senescence were explored. The STING knockdown lentivirus was intra-articularly injected to evaluate its therapeutic impact on OA in mice in vivo. The results showed that the expression of STING was remarkably elevated in the human and mouse OA tissues and in chondrocytes exposed to IL-1β. Overexpression of STING promoted the expression of MMP13, as well as ADAMTS5, but suppressed the expression of Aggrecan, as well as Collagen II; it also enhanced apoptosis and senescence in chondrocytes exposed to and those untreated with IL-1β. The mechanistic study showed that STING activated NF-κB signaling cascade, whereas the blockage of NF-κB signaling attenuated STING-induced apoptosis and senescence, and ameliorated STING-induced ECM metabolism imbalance. In in vivo study, it was demonstrated that STING knockdown alleviated destabilization of the medial meniscus-induced OA development in mice. In conclusion, STING promotes OA by activating the NF-κB signaling cascade, whereas suppression of STING may provide a novel approach for OA therapy.Subject terms: Apoptosis, Senescence     

NF-κB Mediates αvβ3 Integrin-induced Endothelial Cell Survival

The α_vβ₃ integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-κB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and β₃ integrin ligation rapidly increased NF-κB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a β₁-integrin ligand) did not induce NF-κB activity. The α_vβ₃ integrin was most important for osteopontin-mediated NF-κB induction and survival, since adding a neutralizing anti-β₃ integrin antibody blocked NF-κB activity and induced endothelial cell death when cells were plated on osteopontin. NF-κB was required for osteopontin- and vitronectin-induced survival since inhibition of NF-κB activity with nonphosphorylatable IκB completely blocked the protective effect of osteopontin and vitronectin. In contrast, NF-κB was not required for fibronectin, laminin, and collagen type I–induced survival. Activation of NF-κB by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NF-κB reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NF-κB activation. These studies identify NF-κB as an important signaling molecule in α_vβ₃ integrin-mediated endothelial cell survival.     

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.     

The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB signaling

Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5β1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1β, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5β1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1β, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5β1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5β1 as a promising target for treating vascular inflammation in COVID-19.