scholarly journals IKKα plays a major role in canonical NF-kB signalling in colorectal cells

2022 ◽  
Author(s):  
Jack A Prescott ◽  
Kathryn Balmanno ◽  
Jennifer P Mitchell ◽  
Hanneke Okkenhaug ◽  
Simon J Cook
Keyword(s):  
Cell Lines ◽  
Nuclear Translocation ◽  
Transcriptional Response ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Knock Out ◽  
Iκb Kinase ◽  
Sw620 Cells ◽  
Hct116 Cells

Inhibitor of kappa B (IκB) kinase β (IKKβ) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKα being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKα and IKKβ in canonical NF-κB activation in colorectal cells using CRISPR-Cas9 knock-out cell lines, siRNA and selective IKKβ inhibitors. IKKα and IKKβ were redundant for IκBα phosphorylation and turnover since loss of IKKα or IKKβ alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKα was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFα and IL-1α. In these cells IKKβ was far less efficient at compensating for the loss of IKKα than IKKα was able to compensate for the loss of IKKβ. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKβ inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKα loss strongly inhibited TNFα-dependent p65 nuclear translocation, IKKα and IKKβ contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKα in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKβ to date.

scholarly journals Mild Hypothermia Inhibits Nuclear Factor-κB Translocation in Experimental Stroke

2003 ◽  
Vol 23 (5) ◽  
pp. 589-598 ◽  
Author(s):  
Hyung Soo Han ◽  
Murat Karabiyikoglu ◽  
Stephen Kelly ◽  
Raymond A. Sobel ◽  
Midori A. Yenari
Keyword(s):  
Target Genes ◽  
Nuclear Translocation ◽  
Mild Hypothermia ◽  
Brain Temperature ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Cerebral Injury ◽  
Experimental Stroke ◽  
Nuclear Factor ◽  
Iκb Kinase

Nuclear factor-κB (NFκB) is a transcription factor that is activated after cerebral ischemia. NFκB activation leads to the expression of many inflammatory genes involved in the pathogenesis of stroke. The authors previously showed that mild hypothermia is protective even when cooling begins 2 h after stroke onset. In the present study, they examined the influence of hypothermia on NFκB activation. Rats underwent 2 h of transient middle cerebral artery occlusion. Brains were cooled to 33°C immediately after or 2 h after occlusion, and maintained for 2 h. After normothermic ischemia (brain temperature at 38°C), NFκB cytoplasmic expression, nuclear translocation, and binding activity were observed as early as 2 h in the ischemic hemisphere and persisted at 24 h. Hypothermia decreased NFκB translocation and binding activity but did not alter overall expression. Hypothermia also affected the levels of NFκB regulatory proteins by suppressing phosphorylation of NFκB's inhibitory protein (IκB-α) and IκB kinase (IKK-γ) and decreasing IKK activity, but did not alter overall IKK levels. Hypothermia suppressed the expression of two NFκB target genes: inducible nitric oxide synthase and TNF-α. These data suggest that the protective effect of hypothermia on cerebral injury is, in part, related to NFκB inhibition due to decreased activity of IKK.

scholarly journals The viral ankyrin repeat protein (ORF124L) from infectious spleen and kidney necrosis virus attenuates nuclear factor-κB activation and interacts with IκB kinase β

2011 ◽  
Vol 92 (7) ◽  
pp. 1561-1570 ◽  
Author(s):  
Chang-Jun Guo ◽  
Wei-Jian Chen ◽  
Li-Qun Yuan ◽  
Li-Shi Yang ◽  
Shao-Ping Weng ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Mandarin Fish ◽  
Necrosis Virus ◽  
Necrosis Factor Alpha ◽  
Repeat Protein ◽  
Repeat Proteins ◽  
Iκb Kinase

The ankyrin (ANK) repeat is one of the most common protein–protein interaction motifs, found predominantly in eukaryotes and bacteria, but the functions of the ANK repeat are rarely researched in animal viruses, with the exception of poxviruses. Infectious spleen and kidney necrosis virus (ISKNV) is a typical member of the genus Megalocytivirus in the family Iridoviridae and is a causative agent of epizootics in fish. The genome of ISKNV contains four putative viral ANK (vANK) repeat proteins and their functions remain largely unknown. In the present study, it was found that ORF124L, a vANK repeat protein in ISKNV, encodes a protein of 274 aa with three ANK repeats. Transcription of ORF124L was detected at 12 h post-infection (p.i.) and reached a peak at 40 h p.i. ORF124L was found to localize to both the nucleus and the cytoplasm in mandarin fish fry cells. ISKNV ORF124L interacted with the mandarin fish IκB kinase β protein (scIKKβ), and attenuated tumour necrosis factor alpha (TNF-α)- or phorbol myristate acetate (PMA)-induced activity of a nuclear factor κB (NF-κB)–luciferase reporter but did not interfere with the activity of an activator protein 1 (AP-1)–luciferase reporter. Phosphorylation of IκBα and nuclear translocation of NF-κB were also impaired by ISKNV ORF124L. In summary, ORF124L was identified as a vANK repeat protein and its role in inhibition of TNF-α-induced NF-κB signalling was investigated through interaction with the mandarin fish IKKβ. This work may help to improve our understanding of the function of fish iridovirus ANK repeat proteins.

Testes-specific protease 50 (TSP50) promotes cell proliferation through the activation of the nuclear factor κB (NF-κB) signalling pathway

2011 ◽  
Vol 436 (2) ◽  
pp. 457-467 ◽  
Author(s):  
Zhen-Bo Song ◽  
Yong-Li Bao ◽  
Yu Zhang ◽  
Xu-Guang Mi ◽  
Ping Wu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Colony Formation ◽  
Nuclear Translocation ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Nuclear Factor Κb ◽  
Signalling Pathway ◽  
Nuclear Factor ◽  
Iκb Kinase ◽  
Specific Protease

TSP50 (testes-specific protease 50) is a testis-specific expression protein, which is expressed abnormally at high levels in breast cancer tissues. This makes it an attractive molecular marker and a potential target for diagnosis and therapy; however, the biological function of TSP50 is still unclear. In the present study, we show that overexpression of TSP50 in CHO (Chinese-hamster ovary) cells markedly increased cell proliferation and colony formation. Mechanistic studies have revealed that TSP50 can enhance the level of TNFα (tumour necrosis factor α)- and PMA-induced NF-κB (nuclear factor κB)-responsive reporter activity, IκB (inhibitor of NF-κB) α degradation and p65 nuclear translocation. In addition, the knockdown of endogenous TSP50 in MDA-MB-231 cells greatly inhibited NF-κB activity. Co-immunoprecipitation studies demonstrated an interaction of TSP50 with the NF-κB–IκBα complex, but not with the IKK (IκB kinase) α/β–IKKγ complex, which suggested that TSP50, as a novel type of protease, promoted the degradation of IκBα proteins by binding to the NF-κB–IκBα complex. Our results also revealed that TSP50 can enhance the expression of NF-κB target genes involved in cell proliferation. Furthermore, overexpression of a dominant-negative IκB mutant that is resistant to proteasome-mediated degradation significantly reversed TSP50-induced cell proliferation, colony formation and tumour formation in nude mice. Taken together, the results of the present study suggest that TSP50 promotes cell proliferation, at least partially, through activation of the NF-κB signalling pathway.

scholarly journals Delayed apoptosis of human monocytes exposed to immune complexes is reversed by oxaprozin: role of the Akt/IκB kinase/nuclear factor κB pathway

2009 ◽  
Vol 157 (2) ◽  
pp. 294-306 ◽  
Author(s):  
Luciano Ottonello ◽  
Maria Bertolotto ◽  
Fabrizio Montecucco ◽  
Giordano Bianchi ◽  
Franco Dallegri
Keyword(s):  
Immune Complexes ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Human Monocytes ◽  
Iκb Kinase

Abstract 501: Nuclear Factor-κB Is Involved in Platelet CD40 Signaling and Activation

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Ahmed Hachem ◽  
Daniel Yacoub ◽  
Younes Zaid ◽  
Walid Mourad ◽  
Yahye Merhi
Keyword(s):  
P38 Mapk ◽  
Platelet Activation ◽  
Nuclear Translocation ◽  
Kinase Inhibitor ◽  
Tumor Necrosis Factor Receptor ◽  
Nuclear Factor Κb ◽  
Mitogen Activated Protein Kinase ◽  
Nuclear Factor ◽  
Iκb Kinase ◽  
Iκbα Phosphorylation

Introduction and hypothesis: CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets constitute the major source of soluble CD40L (sCD40L), which has been shown to influence platelet activation. We have previously shown that upon ligation, CD40 potentiates platelet activation and aggregation via p38 mitogen activated protein kinase (MAPK) and Rac1 signaling. In B lymphocytes, CD40 induces activation and nuclear translocation of nuclear factor kappa B (NF-κB), which is dependent on the phosphorylation and dissociation of the inhibitor of kappa B α (IκBα). Given that platelets contain NF-κB, we hypothesized that it may be involved in platelet CD40 signaling. Methods and results: In human platelets, sCD40L induced association of tumor necrosis factor receptor associated factor 2 to CD40, and a time-dependant phosphorylation of IκBα, which is indicative of NF-κB activation. Activation of NF-κB in platelets treated with sCD40L was abolished by CD40L blockade. Pretreatment of platelets with the IκBα inhibitor, BAY 11-7082, reversed IκBα phosphorylation induced by sCD40L, without affecting p38 MAPK activation. On the other hand, pretreatment of platelets with the p38 MAPK phosphorylation inhibitor, SB203580, had no effect on IκBα phosphorylation, indicating a divergence in the signaling pathway originating from CD40 upon its ligation. Finally, inhibition of IκBα phosphorylation by either BAY 11-7082 or the IκB kinase inhibitor VII reversed sCD40L induced platelet activation, as measured by P-selectin expression, and the potentiation of platelet aggregation induced by a priming dose of collagen. Conclusion: This study demonstrates the implication of NF-κB in platelet signaling downstream of CD40, where it plays a role in platelet activation and aggregation upon sCD40L stimulation.

scholarly journals Activation of nuclear factor-κB during doxorubicin-induced apoptosis in endothelial cells and myocytes is pro-apoptotic: the role of hydrogen peroxide

2002 ◽  
Vol 367 (3) ◽  
pp. 729-740 ◽  
Author(s):  
Suwei WANG ◽  
Srigiridhar KOTAMRAJU ◽  
Eugene KONOREV ◽  
Shasi KALIVENDI ◽  
Joy JOSEPH ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Apoptotic Pathway ◽  
Toxic Side Effect ◽  
Rat Cardiomyocytes ◽  
A Cell ◽  
Induced Apoptosis

Doxorubicin (DOX) is a widely used anti-tumour drug. Cardiotoxicity is a major toxic side effect of DOX therapy. Although recent studies implicated an apoptotic pathway in DOX-induced cardiotoxicity, the mechanism of DOX-induced apoptosis remains unclear. In the present study, we investigated the role of reactive oxygen species and the nuclear transcription factor nuclear factor κB (NF-κB) during apoptosis induced by DOX in bovine aortic endothelial cells (BAECs) and adult rat cardiomyocytes. DOX-induced NF-κB activation is both dose- and time-dependent, as demonstrated using electrophoretic mobility-shift assay and luciferase and p65 (Rel A) nuclear-translocation assays. Addition of a cell-permeant iron metalloporphyrin significantly suppressed NF-κB activation and apoptosis induced by DOX. Overexpression of glutathione peroxidase, which detoxifies cellular H2O2, significantly decreased DOX-induced NF-κB activation and apoptosis. Inhibition of DOX-induced NF-κB activation by a cell-permeant peptide SN50 that blocks translocation of the NF-κB complex into the nucleus greatly diminished DOX-induced apoptosis. Apoptosis was inhibited when IκB mutant vector, another NF-κB inhibitor, was added to DOX-treated BAECs. These results suggest that NF-κB activation in DOX-treated endothelial cells and myocytes is pro-apoptotic, in contrast with DOX-treated cancer cells, where NF-κB activation is anti-apoptotic. Removal of intracellular H2O2 protects endothelial cells and myocytes from DOX-induced apoptosis, possibly by inhibiting NF-κB activation. These findings suggest a novel mechanism for enhancing the therapeutic efficacy of DOX.

Inhibition of NF-κB essentially contributes to arsenic-induced apoptosis

Blood ◽  
2003 ◽  
Vol 102 (3) ◽  
pp. 1028-1034 ◽  
Author(s):  
Stephan Mathas ◽  
Andreas Lietz ◽  
Martin Janz ◽  
Michael Hinz ◽  
Franziska Jundt ◽  
...  
Keyword(s):  
Cell Lines ◽  
Target Genes ◽  
Nuclear Factor Κb ◽  
Model Systems ◽  
Direct Role ◽  
Iκb Kinase ◽  
Transient Overexpression ◽  
Induced Apoptosis ◽  
Necrosis Factor

Abstract Arsenic can induce apoptosis and is an efficient drug for the treatment of acute promyelocytic leukemia. Currently, clinical studies are investigating arsenic as a therapeutic agent for a variety of malignancies. In this study, Hodgkin/Reed-Sternberg (HRS) cell lines served as model systems to characterize the role of nuclear factor–κB (NF-κB) in arsenic-induced apoptosis. Arsenic rapidly down-regulated constitutive IκB kinase (IKK) as well as NF-κB activity and induced apoptosis in HRS cell lines containing functional IκB proteins. In these cell lines, apoptosis was blocked by inhibition of caspase-8 and caspase-3–like activity. Furthermore, arsenic treatment down-regulated NF-κB target genes, including tumor necrosis factor-αreceptor–associated factor 1 (TRAF1), c-IAP2, interleukin-13 (IL-13), and CCR7. In contrast, cell lines with mutated, functionally inactive IκB proteins or with a weak constitutive IKK/NF-κB activity showed no alteration of the NF-κB activity and were resistant to arsenic-induced apoptosis. A direct role of the NF-κB pathway in arsenic-induced apoptosis is shown by transient overexpression of NF-κB–p65 in L540Cy HRS cells, which protected the cells from arsenic-induced apoptosis. In addition, treatment of NOD/SCID mice with arsenic trioxide induced a dramatic reduction of xenotransplanted L540Cy Hodgkin tumors concomitant with NF-κB inhibition. We conclude that inhibition of NF-κB contributes to arsenic-induced apoptosis. Furthermore, pharmacologic inhibition of the IKK/NF-κB activity might be a powerful treatment option for Hodgkin lymphoma.

scholarly journals MicroRNA-7/NF-κB signaling regulatory feedback circuit regulates gastric carcinogenesis

2015 ◽  
Vol 210 (4) ◽  
pp. 613-627 ◽  
Author(s):  
Xiao-Di Zhao ◽  
Yuan-Yuan Lu ◽  
Hao Guo ◽  
Hua-Hong Xie ◽  
Li-Jie He ◽  
...  
Keyword(s):  
Gene Expression Regulation ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Feedback Circuit ◽  
Poor Survival ◽  
Iκb Kinase ◽  
Genome Wide ◽  
Important Regulator ◽  
Fos Expression

MicroRNAs play essential roles in gene expression regulation during carcinogenesis. Here, we investigated the role of miR-7 and the mechanism by which it is dysregulated in gastric cancer (GC). We used genome-wide screenings and identified RELA and FOS as novel targets of miR-7. Overexpression of miR-7 repressed RELA and FOS expression and prevented GC cell proliferation and tumorigenesis. These effects were clinically relevant, as low miR-7 expression was correlated with high RELA and FOS expression and poor survival in GC patients. Intriguingly, we found that miR-7 indirectly regulated RELA activation by targeting the IκB kinase IKKε. Furthermore, IKKε and RELA can repress miR-7 transcription, which forms a feedback circuit between miR-7 and nuclear factor κB (NF-κB) signaling. Additionally, we demonstrate that down-regulation of miR-7 may occur as a result of the aberrant activation of NF-κB signaling by Helicobacter pylori infection. These findings suggest that miR-7 may serve as an important regulator in GC development and progression.

Mechanism of staurosporine-induced apoptosis in murine hepatocytes

2002 ◽  
Vol 282 (5) ◽  
pp. G825-G834 ◽  
Author(s):  
Guoping Feng ◽  
Neil Kaplowitz
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Jurkat Cells ◽  
Primary Hepatocytes ◽  
Iκb Kinase ◽  
Apoptosis Protein ◽  
Induced Apoptosis

Staurosporine (STS) induces apoptosis in various cell lines. We report in this study that primary cultured mouse hepatocytes are less sensitive to STS compared with Jurkat cells and Huh-7 cells. In contrast to the cell lines, no apparent release of cytochrome c or loss of mitochondrial transmembrane potential was detected in primary hepatocytes undergoing STS-induced apoptosis. Caspase-3 was activated in primary hepatocytes by STS treatment, but caspase-9 and -12 were not activated, and caspase-3 activation is not dependent on caspase-8. These findings point to a novel pathway for caspase-3 activation by STS in primary hepatocytes. Pretreatment with caspase inhibitor converted STS-induced apoptosis of hepatocytes to necrotic cell death without significantly changing total cell death. Thus STS causes hepatocytes to commit to death upstream of the activation of caspases. We also demonstrated that STS dramatically sensitized primary hepatocytes to tumor necrosis factor-α-induced apoptosis. STS activated IκB kinase and nuclear factor-κB (NF-κB) nuclear translocation and DNA binding but inhibited transactivation of IκB-α, inducible nitric oxide synthase, and inhibitor of apoptosis protein-1 in hepatocytes and NF-κB reporter in transfected Huh-7 cells.

scholarly journals DZNep promotes mouse bone defect healing via enhancing both osteogenesis and osteoclastogenesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiankun Cao ◽  
Wenxin He ◽  
Kewei Rong ◽  
Shenggui Xu ◽  
Zhiqian Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Defect ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Cell Counting ◽  
Nuclear Factor ◽  
Defect Healing ◽  
Bone Defect Healing

Abstract Background Enhancer of zeste homolog 2 (EZH2) is a novel oncogene that can specifically trimethylate the histone H3 lysine 27 (H3K27me3) to transcriptionally inhibit the expression of downstream tumor-suppressing genes. As a small molecular inhibitor of EZH2, 3-Deazaneplanocin (DZNep) has been widely studied due to the role of tumor suppression. With the roles of epigenetic regulation of bone cells emerged in past decades, the property and molecular mechanism of DZNep on enhancing osteogenesis had been reported and attracted a great deal of attention recently. This study aims to elucidate the role of DZNep on EZH2-H3K27me3 axis and downstream factors during both osteoclasts and osteoblasts formation and the therapeutic possibility of DZNep on bone defect healing. Methods Bone marrow-derived macrophages (BMMs) cells were cultured, and their responsiveness to DZNep was evaluated by cell counting kit-8, TRAP staining assay, bone resorption assay, podosome actin belt. Bone marrow-derived mesenchymal stem cells (BMSC) were cultured and their responsiveness to DZNep was evaluated by cell counting kit-8, ALP and AR staining assay. The expression of nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), Wnt signaling pathway was determined by qPCR and western blotting. Mouse bone defect models were created, rescued by DZNep injection, and the effectiveness was evaluated by X-ray and micro-CT and histological staining. Results Consistent with the previous study that DZNep enhances osteogenesis via Wnt family member 1(Wnt1), Wnt6, and Wnt10a, our results showed that DZNep also promotes osteoblasts differentiation and mineralization through the EZH2-H3K27me3-Wnt4 axis. Furthermore, we identified that DZNep promoted the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation via facilitating the phosphorylation of IKKα/β, IκB, and subsequently NF-κB nuclear translocation, which credit to the EZH2-H3K27me3-Foxc1 axis. More importantly, the enhanced osteogenesis and osteoclastogenesis result in accelerated mice bone defect healing in vivo. Conclusion DZNep targeting EZH2-H3K27me3 axis facilitated the healing of mice bone defect via simultaneously enhancing osteoclastic bone resorption and promoting osteoblastic bone formation.

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