NF-κB Pathway Contributes to Cadmium-Induced Apoptosis of Porcine Granulosa Cells

To better understand the mechanism of cadmium (Cd)-induced apoptosis of porcine granulosa cells, we examined the nuclear factor-kappa B (NF-κB) p65 subunits intracellular translocation and the expression of some downstream apoptotic-related genes. Apoptosis and reactive oxygen species (ROS) production in porcine granulosa cells exposed to cadmium chloride (CdCl₂) were determined by acridine orange/ethidium bromide double staining and 2,7-dichlorodihydro-fluorescein-diacetate oxidation staining, respectively. The results showed that the apoptosis of porcine granulosa cells induced by CdCl₂ significantly increased in a time- and dose-dependent manner along with the increasing of ROS production, and 10 μM parthenolide, an inhibitor NF-κB, can accelerate the process of apoptosis. Moreover, immunofluorescence and western blot results showed that CdCl₂ could stimulate the translocation of p65 into nucleus in porcine granulosa cells. Furthermore, CdCl₂ also significantly stimulate the expression of Bcl-2 proteins in porcine granulosa cells than that in the control. In contrast, we did not find any change of Bax expression in granulosa cells upon exposure of cadmium. Taken together, these results demonstrate that the activation of NF-κB pathway may play a crucial role in cadmium-induced apoptosis of porcine granulosa cells.     

Irradiation leads to sensitization of hepatocytes to TNF-α-mediated apoptosis by upregulation of IκB expression

This study aimed to reveal the pathophysiological signalling responsible for radiation-induced sensitization of hepatocytes to TNF-α-mediated apoptosis. IκB was upregulated in irradiated hepatocytes. Administration of IκB antisense oligonucleotides prior to irradiation inhibited occurrence of apoptosis after TNF-α administration. Caspases-8, -9 and -3 activities were increased in irradiated hepatocytes and downregulation of apoptosis by IκB antisense oligonucleotides was mediated by suppression of caspases-9 and -3 activation but not of caspase-8 activation, suggesting that radiation-induced sensitization of hepatocytes to TNF-α-mediated apoptosis additionally requires changes upstream of caspase-8 activation. Herein, upregulation of FLIP may play a crucial role. Cleavage of bid, upregulation of bax, downregulation of bcl-2 and release of cytochrome c after TNF-α-administration depend on radiation-induced upregulation of IκB, thus demonstrating an apoptosis permitting effect of IκB. H. Gürleyen and H. Christiansen contributed equally to this work.     

Phosphoinositide 3-kinase activity leads to silica-induced NF-κB activation through interacting with tyrosine-phosphorylated IκB-α and contributing to tyrosine phosphorylation of p65 NF-κB

The role of the subunits of phosphoinositide (PI) 3-kinase in NF-B activation in silica-stimulated RAW 264.7 cells was investigated. Results indicate that PI3-kinase activity was increased in response to silica. The p85 subunit of PI3-kinase interacted with tyrosine-phosphorylated IB- in silica-stimulated cells. PI3-kinase specific inhibitors, such as wortmannin and LY294003, substantially blocked both silica-induced PI3-kinase and NF-B activation. The inhibition of NF-B activation by PI3-kinase inhibitors was also observed in pervanadate-stimulated but not in LPS-stimulated cells. Furthermore, tyrosine phosphorylation of NF-B p65 was enhanced in cells stimulated with silica, pervanadate or LPS, and wortmannin substantially inhibited the phosphorylation event induced by the first two stimulants but not LPS. Antioxidants, such as superoxide dismutase (SOD), N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), blocked silica-induced PI3-kinase activation, suggesting that reactive oxygen species may be important regulatory molecules in NF-B activation by mediating PI3-kinase activation. Our data suggest that p85 and p110 subunits of PI3-kinase play a role in NF-B activation through interaction with tyrosine-phosphorylated IB- and contributing to tyrosine phosphorylation of p65 NF-B.     

Analysis of nuclear factor-κB (NF-κB) essential modulator (NEMO) binding to linear and lysine-linked ubiquitin chains and its role in the activation of NF-κB

Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli.     

Apoptosis of Dedifferentiated Hepatoma Cells is Independent of NF-κB Activation in Response to LPS

Dedifferentiated hepatoma cells, in contrast to most other cell types including hepatoma cells, undergo apoptosis when treated with lipopolysaccharide (LPS) plus the protein synthesis inhibitor cycloheximide (CHx). We recently reported that the dedifferentiated hepatoma cells also exhibit a strong and prolonged NF-κB induction phenotype upon exposure to LPS, suggesting that NF-κB signaling may play a pro-survival role, as reported in several other cell systems. To test the role of NF-κB in preventing LPS-mediated apoptosis, we examined the dedifferentiated cell line M38. Results show that antioxidants strongly inhibited LPS + CHx-mediated cell death in the M38 cells, yet only modestly inhibited NF-κB induction. In addition, inhibition of NF-κB translocation by infection of the M38 cells with an adenoviral vector expressing an IκBα super-repressor did not result in LPS-mediated cell death. These results suggest that unlike TNFα induction, the cell survival pathway activated in response to LPS is independent of NF-κB translocation in the dedifferentiated cells. Addition of inhibitors of JNK, p38 and ERK pathways also failed to elicit LPS-mediated apoptosis similar to that observed when protein synthesis is prevented. Thus, cell survival pathways other than those involving NF-κB inducible gene expression or other well-known pathways appear to be involved in protecting the dedifferentiated hepatoma variant cells from LPS-mediated apoptosis. Importantly, this pro-apoptotic function of LPS appears to be a function of loss of hepatic gene expression, as the parental hepatoma cells resist LPS-mediated apoptosis in the presence of protein synthesis inhibitors.     

Activation of NF-κB by alloferon through down-regulation of antioxidant proteins and IκBα

Alloferon is a 13-amino acid peptide isolated from the bacteria-challenged larvae of the blow fly Calliphora vicina. The pharmaceutical value of the peptide has been well demonstrated by its capacity to stimulate NK cytotoxic activity and interferon (IFN) synthesis in animal and human models, as well as to enhance antiviral and antitumor activities in mice. Antiviral and the immunomodulatory effectiveness of alloferon have also been supported clinically proved in patients suffering with herpes simplex virus (HSV) and human papilloma virus (HPV) infections. To elucidate molecular response to alloferon treatment, we initially screened a model cell line in which alloferon enhanced IFN synthesis upon viral infection. Among the cell lines tested, Namalva was chosen for further proteomic analysis. Fluorescence difference gel electrophoresis (DIGE) revealed that the levels of a series of antioxidant proteins decreased after alloferon treatment, while at least three glycolytic enzymes and four heat-shock proteins were increased in their expression levels. Based on the result of our proteomic analysis, we speculated that alloferon may activate the NF-kappaB signaling pathway. IkappaB kinase (IKK) assay, Western blot analysis on IkappaBalpha and its phosphorylated form at Ser 32, and an NF-kappaB reporter assay verified our proteomics-driven hypothesis. Thus, our results suggest that alloferon potentiates immune cells by activating the NF-kappaB signaling pathway through regulation of redox potential. Since NF-kappaB activation is involved in IFN synthesis, our results provide further clues as to how the alloferon peptide may stimulate IFN synthesis.     

Beyond NF-κB activation: nuclear functions of IκB kinase α

IκB kinase (IKK) complex, the master kinase for NF-κB activation, contains two kinase subunits, IKKα and IKKβ. In addition to mediating NF-κB signaling by phosphorylating IκB proteins during inflammatory and immune responses, the activation of the IKK complex also responds to various stimuli to regulate diverse functions independently of NF-κB. Although these two kinases share structural and biochemical similarities, different sub-cellular localization and phosphorylation targets between IKKα and IKKβ account for their distinct physiological and pathological roles. While IKKβ is predominantly cytoplasmic, IKKα has been found to shuttle between the cytoplasm and the nucleus. The nuclear-specific roles of IKKα have brought increasing complexity to its biological function. This review highlights major advances in the studies of the nuclear functions of IKKα and the mechanisms of IKKα nuclear translocation. Understanding the nuclear activity is essential for targeting IKKα for therapeutics.