PANoptosis represents a newly identified form of programmed cell death that plays a significant role in the autoimmune diseases. Rheumatoid arthritis (RA) is characterized by the presence of autoantibodies. Nevertheless, the specific biomarkers and molecular mechanisms responsible for the apoptotic characteristics of RA remain largely uninvestigated.

We utilized 8 synovial tissue RA datasets. We selected genes associated with PANoptosis from the GeneCard database. By employing the limma, WGCNA, and machine learning algorithms we identified core genes. We utilized consensus clustering analysis to identify distinct PANoptosis subtypes of RA. Boruta algorithm was employed to construct a PANoptosis signature score. The sensitivity of distinct subtypes to drug treatment was verified using an independent dataset.

The SPP1 emerged as the significant gene, with its elevated expression in RA patients. We identified two PANoptosis RA subtypes. Cluster 1 showed high expression of Tregs, resting dendritic cells, and resting mast cells. Cluster 2 exhibited high expression of CD4 memory T cells and follicular helper T cells. Cluster 2 exhibited a higher degree of sensitivity towards immune checkpoint therapy. Employing the Boruta algorithm, a subtype score was devised for 37 PANoptosis genes, successfully discerning the subtypes (AUC = 0.794), wherein patients with elevated scores demonstrated enhanced responsiveness to Rituximab treatment.

Our analysis revealed that SPP1 holds potential biomarker for the diagnosis of RA. Cluster 2 exhibited enhanced sensitivity to immune checkpoint therapy, higher PANoptosis scores, and improved responsiveness to drug treatment. This study offers potential implications in the realm of diagnosis and treatment.

Hypertension and bronchial asthma are a major issue for people's health. As of 2014, approximately one billion adults, or ~ 22% of the world population, have had hypertension. As of 2011, 235-330 million people globally have been affected by asthma and approximately 250,000-345,000 people have died each year from the disease. The development of the effective treatment therapies against these diseases is complicated by their comorbidity features. This is often a major problem in diagnosis and their treatment. Hence, in this study the bioinformatical methodology for the analysis of the comorbidity of these two diseases have been developed. As such, the search for candidate genes related to the comorbid conditions of asthma and hypertension can help in elucidating the molecular mechanisms underlying the comorbid condition of these two diseases, and can also be useful for genotyping and identifying new drug targets.

Using ANDSystem, the reconstruction and analysis of gene networks associated with asthma and hypertension was carried out. The gene network of asthma included 755 genes/proteins and 62,603 interactions, while the gene network of hypertension - 713 genes/proteins and 45,479 interactions. Two hundred and five genes/proteins and 9638 interactions were shared between asthma and hypertension. An approach for ranking genes implicated in the comorbid condition of two diseases was proposed. The approach is based on nine criteria for ranking genes by their importance, including standard methods of gene prioritization (Endeavor, ToppGene) as well as original criteria that take into account the characteristics of an associative gene network and the presence of known polymorphisms in the analysed genes. According to the proposed approach, the genes IL10, TLR4, and CAT had the highest priority in the development of comorbidity of these two diseases. Additionally, it was revealed that the list of top genes is enriched with apoptotic genes and genes involved in biological processes related to the functioning of central nervous system.

The application of methods of reconstruction and analysis of gene networks is a productive tool for studying the molecular mechanisms of comorbid conditions. The method put forth to rank genes by their importance to the comorbid condition of asthma and hypertension was employed that resulted in prediction of 10 genes, playing the key role in the development of the comorbid condition. The results can be utilised to plan experiments for identification of novel candidate genes along with searching for novel pharmacological targets.

The research is aimed to explore the distinct molecular signatures in discriminating the rheumatoid arthritis patients with traditional Chinese medicine (TCM) cold pattern and heat pattern. Twenty patients with typical TCM cold pattern and heat pattern were included. Microarray technology was used to reveal gene expression profiles in CD4+ T cells. The signal intensity of each expressed gene was globally normalized using the R statistics program. The ratio of cold pattern to heat pattern in patients with RA at more or less than 1:2 was taken as the differential gene expression criteria. Protein-protein interaction information for these genes from databases was searched, and the highly connected regions were detected by IPCA algorithm. The significant pathways were extracted from these subnetworks by Biological Network Gene Ontology tool. Twenty-nine genes differentially regulated between cold pattern and heat pattern were found. Among them, 7 genes were expressed significantly more in cold pattern. Biological network of protein-protein interaction information for these significant genes were searched and four highly connected regions were detected by IPCA algorithm to infer significant complexes or pathways in the biological network. Particularly, the cold pattern was related to Toll-like receptor signaling pathway. The following related pathways in heat pattern were included: Calcium signaling pathway; cell adhesion molecules; PPAR signaling pathway; fatty acid metabolism. These results suggest that better knowledge of the main biological processes involved at a given pattern in TCM might help to choose the most appropriate treatment.

The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-kappaB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-kappaB activity. In this study, we have demonstrated the effect of PKA on NF-kappaB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-kappaB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-kappaB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-kappaB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-kappaB activation; PKA-activating agents enhanced NF-kappaB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-kappaB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-kappaB-induced breast cancer cell proliferation and multiple NF-kappaB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-kappaB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies.

Pancreatic cancer is one of the leading causes of cancer mortality in the United States. Current therapy for pancreatic cancer involves surgery, chemotherapy, and radiation therapy; however, the 5-year survival rate remains less than 5%. New strategies for treating pancreatic cancer include targeting intracellular signaling that provides survival advantages to cancer cells. One of these targets is the transcription factor nuclear factor (NF) kappaB, which is activated by a variety of mechanisms. Data demonstrate that increased NF-kappaB activity can promote growth and tumorigenesis, inhibit apoptosis, promote angiogenesis, promote invasion and metastasis, and promote chemoresistance in pancreatic cancer. This review explores the roles of NF-JB in these processes and examines the evidence that different NF-kappaB-inhibiting drugs can improve the treatment of pancreatic cancer.

The p53 binding protein 2 (53BP2) has been identified independently as the interacting protein to p53, Bcl-2, and p65 subunit of nuclear factor kappaB (NF-kappaB). It was demonstrated that over-expression of 53BP2 (renamed as 53BP2S) induces apoptotic cell death. In this study we explored the effect of NF-kappaB activation elicited by a physiological NF-kappaB inducer, interleukin-1beta (IL-1beta), and anti-apoptotic Bcl-2 family proteins on the 53BP2S-mediated apoptosis. We found that both NF-kappaB activation and Bcl-2 family proteins could prevent the 53BP2S-mediated depression of mitochondrial transmembrane potential, activation of caspase-9, cleavage of poly ADP ribose polymerase (PARP), and cell death. These observations suggested that 53BP2S/Bbp and its directly or indirectly interacting proteins might play crucial roles in the regulation of apoptosis and contribute to carcinogenesis. It is also suggested that 53BP2S/Bbp induces apoptosis through the mitochondrial death pathway presumably by counteracting the actions of anti-apoptotic Bcl-2 family proteins. The regulatory network of the 53BP2S-mediated apoptosis cascade including its interacting proteins is discussed.

Involvement of nuclear factor-kappaB (NF-kappaB) in cell survival and proliferation of multiple myeloma has been well established. In this study we observed that NF-kappaB is constitutively activated in all human myeloma cell lines, thus confirming the previous studies. In addition, we found the phosphorylation of p65 subunit of NF-kappaB in addition to the phosphorylation of IkappaBalpha and the activation of NF-kappaB DNA binding and that various target genes of NF-kappaB including bcl-x(L), XIAP, c-IAP1, cyclin D1, and IL-6 are up-regulated. We then examined the effect of a novel IkappaB kinase inhibitor, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile (ACHP). When myeloma cells were treated with ACHP, the cell growth was efficiently inhibited with IC(50) values ranging from 18 to 35 mumol/L concomitantly with inhibition of the phosphorylation of IkappaBalpha/p65 and NF-kappaB DNA-binding, down-regulation of the NF-kappaB target genes, and induction of apoptosis. In addition, we observed the treatment of ACHP augmented the cytotoxic effects of vincristine and melphalan (l-phenylalanine mustard), conventional antimyeloma drugs. These findings indicate that IkappaB kinase inhibitors such as ACHP can sensitize myeloma cells to the cytotoxic effects of chemotherapeutic agents by blocking the antiapoptotic nature of myeloma cells endowed by the constitutive activation of NF-kappaB.

A functional fluorescent neurokinin NK2 receptor, EGFP-NK2, was previously used to follow, by fluorescence resonance energy transfer measurements in living cells, the binding of its fluorescently labeled agonist, bodipy-neurokinin A (NKA). Local agonist application suggested that the activation and desensitization of the NK2 receptors were compartmentalized at the level of the plasma membrane. In this study, fluorescence recovery after photobleaching experiments are carried out at variable observation radius (vrFRAP) to probe EGFP-NK2 receptor mobility and confinement. Experiments are carried out at 20 degrees C to maintain the number of receptors constant at the cell surface during recordings. In the absence of agonist, 35% EGFP-NK2 receptors diffuse within domains of 420 +/- 80 nm in radius with the remaining 65% of receptors able to diffuse with a long range lateral diffusion coefficient between the domains. When cells are incubated with a saturating concentration of NKA, 30% EGFP-NK2 receptors become immobilized in small domains characterized by a radius equal to 170 +/- 50 nm. Biochemical experiments show that the confinement of EGFP-NK2 receptor is not due to its association with rafts at any given time. Colocalization of the receptor with beta-arrestin and transferrin supports that the small domains, containing 30% of activated EGFP-NK2, correspond to clathrin-coated pre-pits. The similar amount of confined EGFP-NK2 receptors found before and after activation (30-35%) is discussed in term of putative transient interactions of the receptors with preexisting scaffolds of signaling molecules.

The invasive potential is increased by glial cell line-derived neurotrophic factor (GDNF) in human pancreatic cancer cell lines. We researched whether the signaling pathway activated by GDNF correlates with the nuclear factor-kappaB (NF-kappaB) in human pancreatic cancer cell lines and whether the inhibition of NF-kappaB activity is associated with suppression of invasive potential.

Proliferation of human pancreatic cancer cell lines (BxPC-3 and MIA PaCa-2) was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays (MTT assay). NF-kappaB activity was examined by dual luciferase assay and electrophoretic mobility shift assay. In addition, to investigate the invasive potential, an in vitro invasion assay was performed.

Proliferation of both cell lines was decreased by a proteasome inhibitor, MG132, in a dose-dependent manner, but proliferation of control and IkappaBalphaM vector-transfected BxPC-3 cells was similar. The invasion cell number and the NF-kappaB activity were increased by GDNF stimulation. However, in the presence of MG132 or IkappaBalphaM, which blocks the nuclear localization of NF-kappaB, both were significantly suppressed. Furthermore, reduced activity of both remained unchanged by GDNF stimulation.

These results indicate that GDNF promotes NF-kappaB activation and that the latter is involved in the invasive potential of human pancreatic cancer cells.

Rheumatoid arthritis (RA) is characterized by progressive inflammation associated with abberrant proliferation of synoviocytes. In order to explore the characteristics of rheumatoid synovial fibroblasts (RSF), we performed the comparative gene expression profile analysis between RSF and normal synovial fibroblasts (NSF) upon tumor necrosis factor (TNF) stimulation. As an initial screening for the genes preferentially induced by TNF in RSF compared with NSF, we have adopted a cDNA array containing well-defined sets of genes responsible for cell growth, cell fate determination, and cellular invasiveness. Differentially expressed genes of interest were confirmed using real-time RT-PCR. We found that TNF induced the expression of Notch-1, Notch-4, and Jagged-2 in RSF. The expression of these proteins was detected in the RA synovial tissues. The nucleus of RA synoviocytes showed strong staining with anti-Notch-1 and Notch-4 antibody. TNF induced the nuclear translocation of Notch intracellular domain in RSF, indicating the elicitation of the Notch signaling. Notch-1, Notch-4, and Jagged-2 proteins were also detected in the developing synovium of mouse embryo. Thus, RSF may have re-acquired the primordial phenotype, accounting for the hyperproliferation and aggressive invasiveness, exhibiting tumor-like phenotype.

Proteins belonging to the nuclear factor kappaB (NF-kappaB) superfamily are involved in osteoclast formation, playing a very important role for both differentiation of osteoclast precursors and survival of mature osteoclasts. Several drugs used to fight bone loss in a variety of human pathologies, including osteoporosis, act by increasing the frequency of osteoclast apoptosis, since it was demonstrated that small changes in osteoclast apoptosis can result in large changes in bone formation. In this respect, targeting of NF-kappaB transcription factor could be of great interest. Among nonviral gene therapy strategies recently proposed to inhibit or even block NF-kappaB activity, the transcription factor decoy (TFD) should be taken in great consideration. The main issue of the present study was to examine the effects of decoy DNA/DNA molecules targeting NF-kappaB on apoptosis of human osteoclasts (OCs), with the aim to interfere with the pathway regulating osteoclast differentiation and programmed cell death. To this aim, we used a mixture of receptor activator of NF-kappaB ligand (RANKL), macrophage colony-stimulating factor (M-CSF) and parathyroid hormone (PTH) to prepare human OCs from peripheral blood cells. Then, transfection with the decoy molecules targeting NF-kappaB was performed. The results obtained demonstrate that in primary cells expressing typical osteoclast markers such as TRAP and MMP9, NF-kappaB decoy significantly stimulated apoptosis. Inhibition of IL-6 expression and induction of Caspase 3 were found in OCs treated with NF-kappaB DNA/DNA decoys. We consider these data as the basis for setting up experimental conditions allowing nonviral gene therapy of several bone disorders.

In this study, a novel interactor of the p65 subunit (RelA) of NF-kappaB has been explored by performing yeast two-hybrid screen using the transactivation domain (TAD) of p65 located in the C terminus as bait. We have isolated a RING finger motif-containing protein, AO7, previously identified as an interacting protein with a ubiquitin-conjugating enzyme, Ubc5B. We confirmed the protein-protein interaction between p65 and AO7 in vitro and in vivo and found that the C-terminal region of AO7 is responsible for the interaction with p65 TAD. AO7 was predominantly localized in the nucleus and activated the NF-kappaB-dependent gene expression upon stimulation with IL-1beta or TNF or overexpression of NF-kappaB-inducing kinase. We found that both the RING finger and the C-terminal regions of AO7 were necessary for the transcriptional activation. When cotransfected with plasmids expressing Gal4-p65 fusion proteins containing various functional domains of p65, we found that p65 TAD was essential for the transcriptional activation mediated by AO7. Furthermore, the p65-mediated transactivation was suppressed by a ubiquitination-defective AO7 mutant in which the essential Cys residue within the RING finger motif was substituted by Ser. These data suggest that AO7 interacts with the p65 TAD and modulates its transcriptional activity.

Transcription factor NF-kappaB controls the expression of a number of genes including those for cell adhesion molecules such as E-selectin, ICAM-1 and VCAM-1. These cell adhesion molecules are known to play important roles in a critical step of tumor metastasis; the arrest of tumor cells on the venous or capillary bed of the target organ. NF-kappaB is activated by extracellular signals such as those elicited by the proinflammatory cytokines, TNF and IL-1. Here we demonstrate that IL-1beta induces nuclear translocation of NF-kappaB in human umbilical vein endothelial cells (HUVEC) followed by induction of cell surface expression of E-selectin, ICAM-1 and VCAM-1, and subsequently augments adhesion of cancer cells expressing sialyl Lewis antigen, a ligand of E-selectin. We also demonstrated that the adhesion of tumor cells to IL-1beta-treated HUVEC was inhibited by gold compounds such as aurothioglucose and aurothiomalate. These observations indicate the involvement of NF-kappaB in cancer metastasis and suggest the feasibility of using gold compounds to prevent metastasis.

We previously reported synergistic induction of apoptosis by IFN-gamma plus either cyclosporin A (CsA) or tacrolimus (FK506) in gastric carcinoma cells. In this study, we aimed to elucidate the mechanism for this synergistic induction of apoptosis. IFN-gamma plus CsA synergistically induced caspase-3 mediated apoptosis in gastric carcinoma cells. Although IFN-gamma induced activation of signal transducer and activator of transcription1 (STAT1) and expression of interferon regulatory factor-1 (IRF-1) mRNA, IFN-gamma alone was not able to induce caspase-3 activation and apoptosis. When gastric carcinoma cells were treated with cyclohexamide, a protein synthesis inhibitor, following IFN-gamma pretreatment, caspase-3 was activated, and apoptosis was markedly induced. These findings suggest the existence of IFN-gamma-induced anti-apoptotic pathway and we evaluated the effect of IFN-gamma and CsA on calcium-sensitive nuclear factor-kappa B (NF-kappa B) activation. IFN-gamma increased intracellular calcium ion concentration ([Ca(2+)](i)) consisting of a spike and a sustained phase, and the latter was completely abrogated by CsA. Activation of NF-kappa B occurred in response to IFN-gamma, and which was markedly inhibited by either CsA or FK506. NF-kappa B decoy also enhanced the cytotoxic effect of IFN-gamma. These results suggest that IFN-gamma may simultaneously induce the STAT1-mediated apoptotic pathway and the anti-apoptotic pathway through calcium-activated NF-kappa B and that inhibition of the latter by CsA may result in dominance of the apoptosis-inducing pathway.

NF-kappaB-inducing kinase (NIK) has been shown to play an essential role in the NF-kappaB activation cascade elicited by lymphotoxin beta receptor (LTbetaR) signaling. However, the molecular mechanism of this pathway remains unclear. In this report we demonstrate that both NIK and IkappaB kinase alpha (IKKalpha) are involved in LTbetaR signaling and that the phosphorylation of the p65 subunit at serine 536 in its transactivation domain 1 (TA1) plays an essential role. We also found that NF-kappaB could be activated in the LTbetaR pathway without altering the level of the phosphorylation of IkappaB and nuclear localization of p65. By using a heterologous transactivation system in which Gal4-dependent reporter gene is activated by the Gal4 DNA-binding domain in fusion with various portions of p65, we found that TA1 serves as a direct target in the NIK-IKKalpha pathway. In addition, mutation studies have revealed the essential role of Ser-536 within TA1 of p65 in transcriptional control mediated by NIK-IKKalpha. Furthermore, we found that Ser-536 was phosphorylated following the stimulation of LTbetaR, and this phosphorylation was inhibited by the kinase-dead dominant-negative mutant of either NIK or IKKalpha. These observations provide evidence for a crucial role of the NIK-IKKalpha cascade for NF-kappaB activation in LTbetaR signaling.

To investigate whether apoptosis is associated with cell adhesion in bronchial epithelium, and whether it contributes to the kinetics of injury and repair of surface epithelia, this study was performed for E-cadherin expression by using immunohistochemistry technique and for apoptosis by TUNEL method. An animal model of smoking was used for this study. The results showed that epithelial cells with membrane anchored E-cadherin decreased remarkably at several time points during 6 months of exposure to smoke (P < 0.01) and then restored to normal level. This fluctuation was associated exclusively with the alteration in number of apoptotic cells (P < 0.01). There was no significant difference in activation of nuclear transcription factor NF-kappa B among groups (P > 0.05). All these suggested that apoptosis is associated with E-cadherin expression in bronchial epithelium of smoking mouse.

Cdc25 activates maturation promoting factor (MPF) and promotes mitosis by removing the inhibitory phosphate from the Tyr-15 of Cdc2 in human cells. In this study, we searched the interacting protein(s) of human Cdc25C using the yeast two-hybrid screen and identified proliferating cell nuclear antigen (PCNA) as an interacting partner of Cdc25C. The interaction between Cdc25C and PCNA was confirmed in vitro and in vivo. Co-immunoprecipitation analyses using human T cell line, Jurkat, further revealed that Cdc25C interacted with PCNA transiently when cells began to enter mitosis. Immunofluorescence analysis also showed that Cdc25C and PCNA were transiently co-localized in the nucleus at the beginning of M phase. Together with the previous observations of the interaction between various cdc/cyclin and PCNA, our findings strongly suggested a potential role of PCNA at the G2 to M phase transition of cell cycle.

Apoptosis is a mode of cell death that plays an important role in both pathological and physiological processes. Research during the last decade has delineated the entire machinery needed for cell death, and its constituents were found to pre-exist in cells. The apoptotic cascade is triggered when cells are exposed to an apoptotic stimulus. It has been known for several years that inhibitors of protein synthesis can potentiate apoptosis that is induced by cytokines and other inducers. Until 1996, it was not understood why protein synthesis inhibitors potentiate apoptosis. Then three reports appeared that suggested the role of the transcription factor NF-kappaB activation in protecting the cells from TNF-induced apoptosis. Since then several proteins have been identified that are regulated by NF-kappaB and are involved in cell survival, proliferation, and protection from apoptosis. It now seems that when a cell is attacked by an apoptotic stimulus, the cell responds first by activating anti-apoptotic mechanisms, which may or may not be followed by apoptosis. Whether or not a cell undergoes proliferation, the survival, or apoptosis, appears to involve a balance between the two mechanisms. Inhibitors of protein synthesis seem to suppress the appearance of protein that are involved in anti-apoptosis. The present review discusses how NF-kappaB controls apoptosis.

Akt is involved in different cellular processes such as cell growth, cell differentiation, and anti-apoptosis.

To investigate the role of Akt in cell growth and survival in PANC-1 pancreatic cancer cells.

Insulin-like growth factor (IGF)-I induced Akt activation in a dose-dependent manner and stimulated anchorage-dependent and anchorage-independent cell growth of PANC-1 cells. In PANC-1 cells infected with adenovirus vectors carrying kinase-deficient Akt, anchorage-dependent and anchorage-independent cell growth was significantly reduced in the presence or absence of IGF-I compared with cells infected with adenovirus vectors carrying wild-type Akt, although IGF-I significantly stimulated cell growth in both transfected cell lines. Conversely, in PANC-1 cells infected with adenovirus vectors carrying kinase-deficient Akt, typical DNA laddering was undetectable in DNA fragmentation assay, and DNA 3;-OH reactivity was not detected in TUNEL assay. We then examined the role of phosphatidylinositol 3-kinase (PI3-K), an upstream mediator of Akt, on cell survival. In PANC-1 cells infected with adenovirus vector carrying a deletion mutant of the 85-kDa regulatory subunit of PI3-K and in cells treated with PI3-K inhibitor wortmannin, typical DNA laddering was evident in DNA fragmentation assay. In TUNEL assay, nuclear condensation and DNA 3;-OH reactivity was observed in approximately 30% of these cells.

The present results indicate that Akt is implicated in cell growth, but not in survival in PANC-1 cells. These results suggest that there may be an alternative survival signal cascade from PI3-K in PANC-1 cells.

The transcription factor nuclear factor kappa B (NF-kappaB) has been implicated in the regulation of genes mainly involved in inflammation and immune response. We analysed the role of NF-kappaB in signalling pathways induced by the hematopoietic growth factor erythropoietin (EPO). Our data, obtained by electrophoretic mobility shift assays (EMSA) and reporter gene assays, show that the intracellular domain of the EPO receptor (EPOR) transmits signals leading to the activation of NF-kappaB. Studies employing an inhibitor specific for the EPOR-associated tyrosine kinase JAK2 suggest that JAK2-dependent pathways are not involved. The induction of an NF-kappaB-triggered reporter gene construct was inhibited by cotransfection of dominant negative forms of the src kinase Lyn, but not by dominant negative JAK2. Using epidermal growth factor (EGF)/EPOR hybrids containing mutant forms of the EPOR intracellular domain, we were able to further define the critical structures for the induction of NF-kappaB. The data show that although the activity of JAK2 seems to be dispensable, its association to the receptor, as well as the phosphorylation of membrane proximal tyrosine residues, are essential. Furthermore, the functional analysis of different receptor forms revealed a correlation of the abilities to induce NF-kappaB activity and to generate antiapoptotic signals.

The nuclear factor kappaB (NF-kappaB) plays a central role in the regulation of genes implicated in immune responses, inflammatory processes, and apoptotic cell death. The zinc finger protein A20 is a cellular inhibitor of NF-kappaB activation by various stimuli and plays a critical role in terminating NF-kappaB responses. The underlying mechanism for NF-kappaB inhibition by A20 is still unknown. A20 has been shown to interact with several proteins including tumor necrosis factor (TNF) receptor-associated factors 2 and 6, as well as the inhibitory protein of kappaB kinase (IKK) gamma protein. Here we report the cloning and characterization of ABIN-2, a previously unknown protein that binds to the COOH-terminal zinc finger domain of A20. NF-kappaB activation induced by TNF and interleukin-1 is inhibited by overexpression of ABIN-2. The latter also inhibits NF-kappaB activation induced by overexpression of receptor-interacting protein or TNF receptor-associated factor 2. In contrast, NF-kappaB activation by overexpression of IKKbeta or direct activators of the IKK complex, such as Tax, cannot be inhibited by ABIN-2. These results indicate that ABIN-2 interferes with NF-kappaB activation upstream of the IKK complex and that it might contribute to the NF-kappaB-inhibitory function of A20.

The apoptosis related proteins Bax, Bcl-2, and NF-kappaB were analyzed in sanguinarine induced apoptosis and blister cell death (BCD) of K562 erythroleukemia cells and in sanguinarine treated high Bcl-2 expressing JM1 pre-B lymphoblastic cells, utilizing immunofluorescence-flow cytometry. Sanguinarine induced apoptosis of K562 cells was found to have increased Bax expression and decreased NF-kappaB, whereas BCD showed a decrease in Bax expression and an increase in NF-kappaB. In contrast, high Bcl-2 expressing JM1 cells, when exposed to the same concentrations (and duration) of sanguinarine that induced PCD and BCD in K562 cells, failed to show the respective morphologies while showing a concomitant increase in Bcl-2. Results from studies with K562 cells suggest that Bax is pro-apoptotic and also that NF-kappaB activation may be associated with BCD. Results from studies with JM1 cells suggest that Bcl-2 is anti-apoptotic and anti-BCD. Results from JM1 cells strengthen the assumption in the literature of the central role Bcl-2 plays in chemoresistance by assuming an anti-PCD role. These results also suggest that, in JM1 cells, Bcl-2 may further complicate chemoresistance by being anti-BCD in nature, in addition to its anti-PCD role.

Heart-specific inhibition of survival pathway gp130 was recently shown to sensitize transgenic mice towards stress stimuli, resulting in rapid onset of cardiac dilatation and heart failure. In order to identify further survival pathways we evaluated the role of transcription factor nuclear factor-kappa B (NF-kappa B) in tumour necrosis factor-alpha (TNF-alpha)-induced apoptosis of cardiomyocytes. TNF-alpha stimulation (10 ng/ml) of both H9c2 cells and primary cardiomyocytes isolated from neonatal Wistar rats resulted in rapid nuclear translocation of NF-kappa B complexes. The NF-kappa B complexes consisted of rel-proteins p50 and p65, as revealed by supershift analysis. Addition of proteasome inhibitor MG132 or adenoviral expression of a truncated I kappa B alpha (I kappa B Delta N) inhibited TNF-alpha-induced NF-kappa B nuclear translocation in a dose-dependent manner. Both neonatal cardiomyocytes and H9c2 cells were resistant to TNF-induced apoptosis. However, specific inhibition of NF-kappa B activation by Ad5-I kappa B alpha Delta N (MOI=50) or MG132 (5 microm) increased apoptosis as measured by subG1-assay (H9c2 cells) and annexin V binding/propidium iodide (neonatal cardiomyocytes, FACS-analysis: 7+/-2% to 26+/-5% annexin V positive/PI negative), respectively. TUNEL-assay double-stained with anti-alpha-sarcomeric actin confirmed apoptosis of neonatal cardiomyocytes. Furthermore, caspase-3 activation was increased by 52+/-7% in neonatal cardiomyocytes after TNF alpha+Ad5-I kappa B alpha Delta N compared to TNF alpha+Ad5-control treatment. Protein levels of hiAP1, hiAP2, x-iAP, bcl-2 and bcl-x(L) were neither downregulated by NF-kappa B inhibition nor upregulated by TNF-alpha stimulation. In summary, cardiomyocytes utilize transcription factor NF-kappa B to activate survival factors in the context of TNF-alpha stimulation. As locally increased levels of TNF-alpha have been detected in heart failure, NF-kappa B activity is essential for cellular homeostasis in the heart.

In this study, we have demonstrated that translocated in liposarcoma (TLS), also termed FUS, is an interacting molecule of the p65 (RelA) subunit of the transcription factor nuclear factor kappaB (NF-kappaB) using a yeast two-hybrid screen. We confirmed the interaction between TLS and p65 by the pull-down assay in vitro and by a coimmunoprecipitation experiment followed by Western blot of the cultured cell in vivo. TLS was originally identified as part of a fusion protein with CHOP arising from chromosomal translocation in human myxoid liposarcomas. TLS has been shown to be involved in TFIID complex formation and associated with RNA polymerase II. However, the role of TLS in transcriptional regulation has not yet been clearly elucidated. We found that TLS enhanced the NF-kappaB-mediated transactivation induced by physiological stimuli such as tumor necrosis factor alpha, interleukin-1beta, and overexpression of NF-kappaB-inducing kinase. TLS augmented NF-kappaB-dependent promoter activity of the intercellular adhesion molecule-1 gene and interferon-beta gene. These results suggest that TLS acts as a coactivator of NF-kappaB and plays a pivotal role in the NF-kappaB-mediated transactivation.