scholarly journals Integrin β3-mediated Src activation regulates apoptosis in IEC-6 cells via Akt and STAT3

2006 ◽  
Vol 397 (3) ◽  
pp. 437-447 ◽  
Author(s):  
Sujoy Bhattacharya ◽  
Ramesh M. Ray ◽  
Leonard R. Johnson
Keyword(s):  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Janus Kinase ◽  
Intestinal Epithelial ◽  
Akt Phosphorylation ◽  
Antiapoptotic Proteins ◽  
Integrin Β3 ◽  
Extracellular Signal ◽  
Src Activation ◽  
Factor Α

Intestinal epithelial (IEC-6) cells are resistant to apoptosis following the inhibition of ODC (ornithine decarboxylase) and subsequent polyamine depletion. The depletion of polyamines rapidly activates NF-κB (nuclear factor κB) and STAT3 (signal transducer and activator of transcription 3), which is responsible for the observed decrease in apoptosis. Since both NF-κB and STAT3 signalling pathways can be activated by Src kinase, we examined its role in the antiapoptotic response. Inhibition of ODC by DFMO (α-difluoromethylornithine) increased the activity of Src and ERK1/2 (extracellular-signal-regulated kinase 1/2) within 30 min, which was prevented by exogenous polyamines added to the DFMO-containing medium. Conversely, epidermal growth factor-mediated Src and ERK1/2 activation was not prevented by the addition of polyamines. Inhibition of Src with PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine} and a DN-Src (dominant-negative Src) construct prevented the activation of Akt, JAK (Janus kinase) and STAT3. Spontaneous apoptosis was increased in DN-Src-expressing cells and the protective effect of polyamine depletion was lost. Polyamine depletion by DFMO increased integrin β3 Tyr785 phosphorylation. Cells plated on fibronectin had significantly higher β3 phosphorylation and Src activation compared with plastic. Exogenous polyamines added to the fibronectin matrix prevented Src activation. Arg-Gly-Asp-Ser inhibited β3, Src and Akt phosphorylation and sensitized polyamine-depleted cells to tumour necrosis factor α/cycloheximide-mediated apoptosis. Fibronectin activated Src and subsequently protected cells from apoptosis. Together, these results suggest that the inhibition of ODC rapidly removes a small pool of available polyamines triggering the activation of β3 integrin, which in turn activates Src. The subsequent Akt and JAK activation is accompanied by translocation of NF-κB and STAT3 to the nucleus and the synthesis of antiapoptotic proteins.

scholarly journals Molecular mechanism mediating enteric bacterial translocation after severe burn: the role of cystic fibrosis transmembrane conductance regulator

2021 ◽  
Vol 9 ◽  
Author(s):  
Xinzhu Liu ◽  
Yu Chen ◽  
Bo You ◽  
Yuan Peng ◽  
Yajie Chen ◽  
...  
Keyword(s):  
Intestinal Barrier ◽  
Nuclear Factor Κb ◽  
Inflammatory Factors ◽  
Intestinal Epithelial ◽  
Severe Burn ◽  
Hypoxic Injury ◽  
Extracellular Signal ◽  
Factor Α

Abstract Background Gut ischemia and hypoxia post severe burn leads to breakdown of intestinal epithelial barrier and enteric bacterial translocation (EBT), resulting in serious complications, such as systemic inflammatory response syndrome, sepsis and multiple organ failure. Cystic fibrosis transmembrane conductance regulator (CFTR) is known to be downregulated by hypoxia and modulate junctional complexes, which are crucial structures maintaining the intestinal barrier. This study aimed to investigate whether CFTR plays a role in both regulating the intestinal barrier and mediating EBT post severe burn, as well as the signaling pathways involved in these processes. Methods An in vitro Caco-2 cell model subjected to hypoxic injury and an in vivo mouse model with a 30% total body surface area full-thickness dermal burn were established. DF 508 mice (mice with F508del CFTR gene mutation) were used as an in vivo model to further demonstrate the role of CFTR in maintaining normal intestinal barrier function. QRT-PCR, western blot, ELISA, TER assay and immunofluorescence staining were used to detect the expression and localization of CFTR and tight junction proteins, as well as the function of tight junctions. Results Our data indicated that, in Caco-2 cells, the hypoxia condition significantly reduced CFTR expression; activated extracellular signal-regulated kinase and nuclear factor-κB signaling; elevated secretion of inflammatory factors (tumor necrosis factor-α, interleukin-1β and interleukin-8); downregulated zonula occludens-1, occludin and E-cadherin expression; decreased transepithelial electrical resistance values; and led to a cellular mislocation of ZO-1. More importantly, knockdown of CFTR caused similar alterations. The upregulation of inflammatory factors and downregulation of tight junction proteins (ZO-1 and occludin) induced by knockdown of CFTR could be reversed by specific extracellular signal-regulated kinase or nuclear factor-κB inhibition. In support of the in vitro data, exuberant secretion of pro-inflammatory mediators and EBT was observed in the intestine of severely burnt mice in vivo. EBT occurred in DF508 mice (mice with the F508del CFTR gene mutation), accompanied by augmented tumor necrosis factor-α, interleukin-1β and interleukin-8 levels in the ileum compared to wildtype mice. In addition, vitamin D3 was shown to protect the intestinal epithelial barrier from hypoxic injury. Conclusions Collectively, the present study illustrated that CFTR and downstream signaling were critical in modulating the intestinal epithelial junction and EBT post severe burn.

Increased cytochrome P-450 2E1 expression sensitizes hepatocytes to c-Jun-mediated cell death from TNF-α

2002 ◽  
Vol 282 (2) ◽  
pp. G257-G266 ◽  
Author(s):  
Hailing Liu ◽  
Brett E. Jones ◽  
Cynthia Bradham ◽  
Mark J. Czaja
Keyword(s):  
Cell Death ◽  
Oxidant Stress ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Tnf Α ◽  
Cyp2e1 Expression ◽  
Factor Α ◽  
Jnk Activation ◽  
Cytochrome P 450 ◽  
Necrosis Factor

The mechanisms underlying hepatocyte sensitization to tumor necrosis factor-α (TNF-α)-mediated cell death remain unclear. Increases in hepatocellular oxidant stress such as those that occur with hepatic overexpression of cytochrome P-450 2E1 (CYP2E1) may promote TNF-α death. TNF-α treatment of hepatocyte cell lines with differential CYP2E1 expression demonstrated that overexpression of CYP2E1 converted the hepatocyte TNF-α response from proliferation to apoptotic and necrotic cell death. Death occurred despite the presence of increased levels of nuclear factor-κB transcriptional activity and was associated with increased lipid peroxidation and GSH depletion. CYP2E1-overexpressing hepatocytes had increased basal and TNF-α-induced levels of c-Jun NH2-terminal kinase (JNK) activity, as well as prolonged JNK activation after TNF-α stimulation. Sensitization to TNF-α-induced cell death by CYP2E1 overexpression was inhibited by antioxidants or adenoviral expression of a dominant-negative c-Jun. Increased CYP2E1 expression sensitized hepatocytes to TNF-α toxicity mediated by c-Jun and overwhelming oxidative stress. The chronic increase in intracellular oxidant stress created by CYP2E1 overexpression may serve as a mechanism by which hepatocytes are sensitized to TNF-α toxicity in liver disease.

scholarly journals PKCζ decreases eNOS protein stability via inhibitory phosphorylation of ERK5

Blood ◽  
2010 ◽  
Vol 116 (11) ◽  
pp. 1971-1979 ◽  
Author(s):  
Patrizia Nigro ◽  
Jun-ichi Abe ◽  
Chang-Hoon Woo ◽  
Kimio Satoh ◽  
Carolyn McClain ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Protein Stability ◽  
Catalytic Domain ◽  
Dominant Negative ◽  
Enos Expression ◽  
Extracellular Signal ◽  
Dominant Negative Form ◽  
Factor Α ◽  
Endothelial Nitric Oxide ◽  
Enos Protein

Abstract PKCζ has emerged as a pathologic mediator of endothelial cell dysfunction, based on its essential role in tumor necrosis factor α (TNFα)–mediated inflammation. In contrast, extracellular signal–regulated kinase 5 (ERK5) function is required for endothelial cell homeostasis as shown by activation of Krüppel-like factor 2 (KLF2), increased endothelial nitric-oxide synthase (eNOS) expression, and inhibition of apoptosis. We hypothesized that protein kinase C ζ (PKCζ) activation by TNFα would inhibit the ERK5/KLF2/eNOS pathway. TNFα inhibited the steady laminar flow–induced eNOS expression, and this effect was reversed by the dominant-negative form of PKCζ (Ad.DN-PKCζ). In addition, ERK5 function was inhibited by either TNFα or the transfection of the catalytic domain of PKCζ. This inhibition was reversed by PKCζ small interfering RNA. PKCζ was found to bind to ERK5 under basal conditions with coimmunoprecipitation and the mammalian 2-hybrid assay. Furthermore, PKCζ phosphorylates ERK5, and mutation analysis showed that the preferred site is S486. Most importantly, we found that the predominant effect of TNFα stimulation of PKCζ was to decrease eNOS protein stability that was recapitulated by transfecting Ad.ERK5S486A mutant. Finally, aortic en face analysis of ERK5/PKCζ activity showed high PKCζ and ERK5 staining in the athero-prone region. Taken together our results show that PKCζ binds and phosphorylates ERK5, thereby decreasing eNOS protein stability and contributing to early events of atherosclerosis.

scholarly journals Cells migrating to sites of tissue damage in response to the danger signal HMGB1 require NF-κB activation

2007 ◽  
Vol 179 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Roberta Palumbo ◽  
Beatriz G. Galvez ◽  
Tobias Pusterla ◽  
Francesco De Marchis ◽  
Giulio Cossu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Tissue Damage ◽  
General Circulation ◽  
High Mobility ◽  
Nuclear Factor Κb ◽  
Bacterial Invasion ◽  
Extracellular Signal ◽  
Factor Α ◽  
Kinase Phosphorylation

Tissue damage is usually followed by healing, as both differentiated and stem cells migrate to replace dead or damaged cells. Mesoangioblasts (vessel-associated stem cells that can repair muscles) and fibroblasts migrate toward soluble factors released by damaged tissue. Two such factors are high mobility group box 1 (HMGB1), a nuclear protein that is released by cells undergoing unscheduled death (necrosis) but not by apoptotic cells, and stromal derived factor (SDF)–1/CXCL12. We find that HMGB1 activates the canonical nuclear factor κB (NF-κB) pathway via extracellular signal-regulated kinase phosphorylation. NF-κB signaling is necessary for chemotaxis toward HMGB1 and SDF-1/CXCL12, but not toward growth factor platelet-derived growth factor, formyl-met-leu-phe (a peptide that mimics bacterial invasion), or the archetypal NF-κB–activating signal tumor necrosis factor α. In dystrophic mice, mesoangioblasts injected into the general circulation ingress inefficiently into muscles if their NF-κB signaling pathway is disabled. These findings suggest that NF-κB signaling controls tissue regeneration in addition to early events in inflammation.

Induction of group IVC phospholipase A2 in allergic asthma: transcriptional regulation by TNFα in bronchoepithelial cells

2012 ◽  
Vol 442 (1) ◽  
pp. 127-137 ◽  
Author(s):  
Justin S. Bickford ◽  
Kimberly J. Newsom ◽  
John-David Herlihy ◽  
Christian Mueller ◽  
Benjamin Keeler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Phospholipase A2 ◽  
Rna Polymerase Ii ◽  
Allergic Asthma ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Lung Epithelial Cells ◽  
Bioactive Lipids ◽  
Eicosanoid Release ◽  
Factor Α

Airway inflammation in allergen-induced asthma is associated with eicosanoid release. These bioactive lipids exhibit anti- and pro-inflammatory activities with relevance to pulmonary pathophysiology. We hypothesized that sensitization/challenge using an extract from the ubiquitous fungus Aspergillus fumigatus in a mouse model of allergic asthma would result in altered phospholipase gene expression, thus modulating the downstream eicosanoid pathway. We observed the most significant induction in the group IVC PLA2 (phospholipase A2) [also known as cPLA2γ (cytosolic PLA2γ) or PLA2G4C]. Our results infer that A. fumigatus extract can induce cPLA2γ levels directly in eosinophils, whereas induction in lung epithelial cells is most likely to be a consequence of TNFα (tumour necrosis factor α) secretion by A. fumigatus-activated macrophages. The mechanism of TNFα-dependent induction of cPLA2γ gene expression was elucidated through a combination of promoter deletions, ChIP (chromatin immunoprecipitation) and overexpression studies in human bronchoepithelial cells, leading to the identification of functionally relevant CRE (cAMP-response element), NF-κB (nuclear factor κB) and E-box promoter elements. ChIP analysis demonstrated that RNA polymerase II, ATF-2 (activating transcription factor 2)–c-Jun, p65–p65 and USF (upstream stimulating factor) 1–USF2 complexes are recruited to the cPLA2γ enhancer/promoter in response to TNFα, with overexpression and dominant-negative studies implying a strong level of co-operation and interplay between these factors. Overall, our results link cytokine-mediated alterations in cPLA2γ gene expression with allergic asthma and outline a complex regulatory mechanism.

Role of the MyD88 transduction signaling pathway in endothelial activation by antiphospholipid antibodies

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3495-3500 ◽  
Author(s):  
Elena Raschi ◽  
Cinzia Testoni ◽  
Daniela Bosisio ◽  
Maria O. Borghi ◽  
Takao Koike ◽  
...  
Keyword(s):  
Antiphospholipid Antibodies ◽  
Fetal Loss ◽  
Endothelial Activation ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Signaling Cascade ◽  
Glycoprotein I ◽  
Tnf Α ◽  
Factor Α

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the persistent presence of antiphospholipid antibodies (aPLs) and recurrent thrombosis or fetal loss. The thrombophilic state has been partially related to the induction of a proinflammatory and procoagulant endothelial cell (EC) phenotype induced by anti–β2-glycoprotein I (β2-GPI) antibodies that bind β2-GPI expressed on the EC surface. Anti–β2-GPI antibody binding has been shown to induce nuclear factor-κB (NF-κB) translocation leading to a proinflammatory EC phenotype similar to that elicited by interaction with microbial products (lipopolysaccharide [LPS]) and proinflammatory cytokines (interleukin 1β [IL-1β], tumor necrosis factor α [TNF-α]). However, the upstream signaling events are not characterized yet. To investigate the endothelial signaling cascade activated by anti–β2-GPI antibodies, we transiently cotransfected immortalized human microvascular endothelial cells (HMEC-1) with dominant-negative constructs of different components of the pathway (ΔTRAF2, ΔTRAF6, ΔMyD88) together with reporter genes (NF-κB luciferase and pCMV-β-galactosidase). Results showed that both human anti–β2-GPI IgM monoclonal antibodies as well as polyclonal affinity-purified anti–β2-GPI IgG display a signaling cascade comparable to that activated by LPS or IL-1. ΔTRAF6 and ΔMyD88 significantly abrogate antibody-induced as well as IL-1– or LPS-induced NF-κB activation, whereas ΔTRAF2 (involved in NF-κB activation by TNF) does not affect it. Moreover, anti– β2-GPI antibodies and LPS followed the same time kinetic of IL-1 receptor–activated kinase (IRAK) phosphorylation, suggesting an involvement of the toll-like receptor (TLR) family. Our findings demonstrate that anti–β2-GPI antibodies react with their antigen likely associated to a member of the TLR/IL-1 receptor family on the EC surface and directly induce activation.

Developmentally regulated tumor necrosis factor-α induced nuclear factor-κB activation in intestinal epithelium

2007 ◽  
Vol 292 (5) ◽  
pp. G1411-G1419 ◽  
Author(s):  
Erika C. Claud ◽  
Xiaoqiong Zhang ◽  
Elaine O. Petrof ◽  
Jun Sun
Keyword(s):  
Tumor Necrosis Factor ◽  
Epithelial Cells ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Α ◽  
Intestinal Epithelial Cells ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Intestinal Epithelial ◽  
Factor Α ◽  
Necrosis Factor

Premature infants are susceptible to many conditions that are inflammatory in nature. For this patient population, which is expecting the intrauterine environment, pathways necessary for fetal life and development may not have completed the transitions necessary for extrauterine life. In this study, responses to tumor necrosis factor-α were compared in human fetal and adult intestinal epithelial cell lines along with preweaned and postweaned mouse intestinal sections to identify a potential developmental difference that may explain the heightened inflammatory response of preterm infants. The nuclear factor-κB (NF-κB) pathway regulates a wide variety of genes involved in immune and inflammatory processes. We report that, compared with adult intestinal epithelial cells, immature intestinal epithelial cells have increased NF-κB activity associated with increased NF-κB-DNA binding and transcriptional activity. This increased activity appears due to inadequate inhibition of signaling leading to NF-κB activation since there is also increased phosphorylation, ubiquitination, and degradation of the inhibitor of NF-κB in conjunction with decreased baseline expression and delayed resynthesis of this inhibitor. Thus we demonstrate a potential mechanism for the heightened inflammatory response of immature intestinal epithelial cells.

Sign in / Sign up

Export Citation Format

Share Document