NF-κB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells

2001 ◽  
Vol 280 (5) ◽  
pp. G992-G1004 ◽  
Author(s):  
Li Li ◽  
Jaladanki N. Rao ◽  
Barbara L. Bass ◽  
Jian-Ying Wang
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Nuclear Translocation ◽  
Intestinal Epithelial Cells ◽  
Apoptotic Cell ◽  
Binding Activity ◽  
Cell Renewal ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Induced Apoptosis

The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G1 phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-κB (NF-κB) in this process. Although depletion of polyamines by α-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-α (TNF-α) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-κB proteins, induced NF-κB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-κB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-α in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-α-induced cell death, 2) polyamine depletion induces NF-κB activation, and 3) disruption of NF-κB function is associated with altered susceptibility to apoptosis induced by STS or TNF-α. These findings suggest that increased NF-κB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.

NF-κB-mediated IAP expression induces resistance of intestinal epithelial cells to apoptosis after polyamine depletion

2004 ◽  
Vol 286 (5) ◽  
pp. C1009-C1018 ◽  
Author(s):  
Tongtong Zou ◽  
Jaladanki N. Rao ◽  
Xin Guo ◽  
Lan Liu ◽  
Huifang M. Zhang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Caspase 3 ◽  
Intestinal Epithelial Cells ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Induced Apoptosis ◽  
Necrosis Factor ◽  
Active Caspase

Apoptosis plays a crucial role in maintenance of intestinal epithelial integrity and is highly regulated by numerous factors, including cellular polyamines. We recently showed that polyamines regulate nuclear factor (NF)-κB activity in normal intestinal epithelial (IEC-6) cells and that polyamine depletion activates NF-κB and promotes resistance to apoptosis. The current study went further to determine whether the inhibitors of apoptosis (IAP) family of proteins, c-IAP2 and XIAP, are downstream targets of activated NF-κB and play a role in antiapoptotic activity of polyamine depletion in IEC-6 cells. Depletion of cellular polyamines by α-difluoromethylornithine not only activated NF-κB activity but also increased expression of c-IAP2 and XIAP. Specific inhibition of NF-κB by the recombinant adenoviral vector containing IκBα superrepressor (Ad Iκ BSR) prevented the induction of c-IAP2 and XIAP in polyamine-deficient cells. Decreased levels of c-IAP2 and XIAP proteins by inactivation of NF-κB through Ad Iκ BSR infection or treatment with the specific inhibitor Smac also overcame the resistance of polyamine-depleted cells to apoptosis induced by the combination of tumor necrosis factor (TNF)-α and cycloheximide (CHX). Although polyamine depletion did not alter levels of procaspase-3 protein, it inhibited formation of the active caspase-3. Decreased levels of c-IAP2 and XIAP by Smac prevented the inhibitory effect of polyamine depletion on the cleavage of procaspase-3 to the active caspase-3. These results indicate that polyamine depletion increases expression of c-IAP2 and XIAP by activating NF-κB in intestinal epithelial cells. Increased c-IAP2 and XIAP after polyamine depletion induce the resistance to TNF-α/CHX-induced apoptosis, at least partially, through inhibition of the caspase-3 activity.

Induced focal adhesion kinase expression suppresses apoptosis by activating NF-κB signaling in intestinal epithelial cells

2006 ◽  
Vol 290 (5) ◽  
pp. C1310-C1320 ◽  
Author(s):  
Huifang M. Zhang ◽  
Kaspar M. Keledjian ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Liu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Intestinal Epithelial Cells ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Induced Apoptosis

Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-κB signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-κB activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF-α plus cycloheximide (TNF-α/CHX). Specific inhibition of NF-κB by the recombinant adenoviral vector containing the IκBα superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-κB activity and increased the sensitivity to TNF-α/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-κB activity and resulted in increased resistance to TNF-α/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-α/CHX-induced apoptosis, at least partially, through the activation of NF-κB signaling in IECs.

scholarly journals Campylobacter jejuni Cas9 Modulates the Transcriptome in Caco-2 Intestinal Epithelial Cells

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1193
Author(s):  
Chinmoy Saha ◽  
Deborah Horst-Kreft ◽  
Inez Kross ◽  
Peter J. van der Spek ◽  
Rogier Louwen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Epithelial Cells ◽  
Campylobacter Jejuni ◽  
Nuclear Translocation ◽  
Intestinal Epithelial Cells ◽  
Nuclear Factor Κb ◽  
Intestinal Epithelial ◽  
Human Intestinal Epithelial Cells ◽  
Cellular Pathways

The zoonotic human pathogen Campylobacter jejuni is known for its ability to induce DNA-damage and cell death pathology in humans. The molecular mechanism behind this phenomenon involves nuclear translocation by Cas9, a nuclease in C. jejuni (CjeCas9) that is the molecular marker of the Type II CRISPR-Cas system. However, it is unknown via which cellular pathways CjeCas9 drives human intestinal epithelial cells into cell death. Here, we show that CjeCas9 released by C. jejuni during the infection of Caco-2 human intestinal epithelial cells directly modulates Caco-2 transcriptomes during the first four hours of infection. Specifically, our results reveal that CjeCas9 activates DNA damage (p53, ATM (Ataxia Telangiectasia Mutated Protein)), pro-inflammatory (NF-κB (Nuclear factor-κB)) signaling and cell death pathways, driving Caco-2 cells infected by wild-type C. jejuni, but not when infected by a cas9 deletion mutant, towards programmed cell death. This work corroborates our previous finding that CjeCas9 is cytotoxic and highlights on a RNA level the basal cellular pathways that are modulated.

TNF-α/cycloheximide-induced apoptosis in intestinal epithelial cells requires Rac1-regulated reactive oxygen species

2008 ◽  
Vol 294 (4) ◽  
pp. G928-G937 ◽  
Author(s):  
Shi Jin ◽  
Ramesh M. Ray ◽  
Leonard R. Johnson
Keyword(s):  
Reactive Oxygen Species ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Ros Production ◽  
Oxygen Species ◽  
Intestinal Epithelial ◽  
Mitochondrial Ros ◽  
Apoptosis Inhibition ◽  
Tnf Α ◽  
Induced Apoptosis

Previously we have shown that both Rac1 and c-Jun NH2-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-α/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-α-induced apoptosis. In this study, we showed that TNF-α/CHX-induced ROS production and hydrogen peroxide (H2O2)-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-α-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-α-induced mitochondrial ROS production. Moreover, TNF-α-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-α-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-α-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells.

scholarly journals Prohibitin Inhibits Tumor Necrosis Factor alpha–induced Nuclear Factor-kappa B Nuclear Translocation via the Novel Mechanism of Decreasing Importin α3 Expression

2009 ◽  
Vol 20 (20) ◽  
pp. 4412-4423 ◽  
Author(s):  
Arianne L. Theiss ◽  
Aaron K. Jenkins ◽  
Ngozi I. Okoro ◽  
Jan-Michael A. Klapproth ◽  
Didier Merlin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Inflammation ◽  
Nuclear Translocation ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Factor Alpha

Expression of prohibitin 1 (PHB), a multifunctional protein in the cell, is decreased during inflammatory bowel disease (IBD). Little is known regarding the regulation and role of PHB during intestinal inflammation. We examined the effect of tumor necrosis factor alpha (TNF-α), a cytokine that plays a central role in the pathogenesis of IBD, on PHB expression and the effect of sustained PHB expression on TNF-α activation of nuclear factor-kappa B (NF-κB) and epithelial barrier dysfunction, two hallmarks of intestinal inflammation. We show that TNF-α decreased PHB protein and mRNA abundance in intestinal epithelial cells in vitro and in colon mucosa in vivo. Sustained expression of prohibitin in intestinal epithelial cells in vitro and in vivo (prohibitin transgenic mice, PHB TG) resulted in a marked decrease in TNF-α–induced nuclear translocation of the NF-κB protein p65, NF-κB/DNA binding, and NF-κB–mediated transcriptional activation despite robust IκB-α phosphorylation and degradation and increased cytosolic p65. Cells overexpressing PHB were protected from TNF-α–induced increased epithelial permeability. Expression of importin α3, a protein involved in p50/p65 nuclear import, was decreased in cells overexpressing PHB and in colon mucosa of PHB TG mice. Restoration of importin α3 levels sustained NF-κB activation by TNF-α during PHB transfection. These results suggest that PHB inhibits NF-κB nuclear translocation via a novel mechanism involving alteration of importin α3 levels. TNF-α decreases PHB expression in intestinal epithelial cells and restoration of PHB expression in these cells can protect against the deleterious effects of TNF-α and NF-κB on barrier function.

scholarly journals TNF-α synergises with IFN-γ to induce caspase-8-JAK1/2-STAT1-dependent death of intestinal epithelial cells

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Jerzy A. Woznicki ◽  
Nisha Saini ◽  
Peter Flood ◽  
Subhasree Rajaram ◽  
Ciaran M. Lee ◽  
...  
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Ex Vivo ◽  
Caspase 8 ◽  
Causative Role ◽  
Intestinal Epithelial ◽  
Cell Death Pathway ◽  
Tnf Α ◽  
Ifn Γ

AbstractRewiring of host cytokine networks is a key feature of inflammatory bowel diseases (IBD) such as Crohn’s disease (CD). Th1-type cytokines—IFN-γ and TNF-α—occupy critical nodes within these networks and both are associated with disruption of gut epithelial barrier function. This may be due to their ability to synergistically trigger the death of intestinal epithelial cells (IECs) via largely unknown mechanisms. In this study, through unbiased kinome RNAi and drug repurposing screens we identified JAK1/2 kinases as the principal and nonredundant drivers of the synergistic killing of human IECs by IFN-γ/TNF-α. Sensitivity to IFN-γ/TNF-α-mediated synergistic IEC death was retained in primary patient-derived intestinal organoids. Dependence on JAK1/2 was confirmed using genetic loss-of-function studies and JAK inhibitors (JAKinibs). Despite the presence of biochemical features consistent with canonical TNFR1-mediated apoptosis and necroptosis, IFN-γ/TNF-α-induced IEC death was independent of RIPK1/3, ZBP1, MLKL or caspase activity. Instead, it involved sustained activation of JAK1/2-STAT1 signalling, which required a nonenzymatic scaffold function of caspase-8 (CASP8). Further modelling in gut mucosal biopsies revealed an intercorrelated induction of the lethal CASP8-JAK1/2-STAT1 module during ex vivo stimulation of T cells. Functional studies in CD-derived organoids using inhibitors of apoptosis, necroptosis and JAKinibs confirmed the causative role of JAK1/2-STAT1 in cytokine-induced death of primary IECs. Collectively, we demonstrate that TNF-α synergises with IFN-γ to kill IECs via the CASP8-JAK1/2-STAT1 module independently of canonical TNFR1 and cell death signalling. This non-canonical cell death pathway may underpin immunopathology driven by IFN-γ/TNF-α in diverse autoinflammatory diseases such as IBD, and its inhibition may contribute to the therapeutic efficacy of anti-TNFs and JAKinibs.

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