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1.  Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. 
Journal of Clinical Investigation  1996;97(7):1715-1722.
Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of genes involved in the inflammatory and proliferative responses of cells. The present study was designed to elucidate the implication of NF-kappaB/Rel in the pathogenesis of atherosclerosis. Activation of the dimeric NF-kappaB complex is regulated at a posttranslational level and requires the release of the inhibitor protein IkappaB. The newly developed mAb alpha-p65mAb recognizes the IkappaB binding region on the p65 (RelA) DNA binding subunit and therefore selectively reacts with p65 in activated NF-kappaB. Using immunofluorescence and immunohistochemical techniques, activated NF-kappaB was detected in the fibrotic-thickened intima/media and atheromatous areas of the atherosclerotic lesion. Activation of NF-kappaB was identified in smooth muscle cells, macrophages, and endothelial cells. Little or no activated NF-kappaB was detected in vessels lacking atherosclerosis. Electrophoretic mobility shift assays and colocalization of activated NF-kappaB with NF-kappaB target gene expression suggest functional implications for this transcription factor in the atherosclerotic lesion. This study demonstrates the presence of activated NF-kappaB in human atherosclerotic tissue for the first time. Atherosclerosis, characterized by features of chronic inflammation and proliferative processes, may be a paradigm for the involvement of NF-kappaB/Rel in chronic inflammatory disease.
PMCID: PMC507236  PMID: 8601637
2.  IkappaBalpha deficiency results in a sustained NF-kappaB response and severe widespread dermatitis in mice. 
Molecular and Cellular Biology  1996;16(5):2341-2349.
The ubiquitous transcription factor NF-kappaB is an essential component in signal transduction pathways, in inflammation, and in the immune response. NF-kappaB is maintained in an inactive state in the cytoplasm by protein-protein interaction with IkappaBalpha. Upon stimulation, rapid degradation of IkappaBalpha allows nuclear translocation of NF-kappaB. To study the importance of IkappaBalpha in signal transduction, IkappaBalpha-deficient mice were derived by gene targeting. Cultured fibroblasts derived from IkappaBalpha-deficient embryos exhibit levels of NF-kappaB1, NF-kappaB2, RelA, c-Rel, and IkappaBbeta similar to those of wild-type fibroblasts. A failure to increase nuclear levels of NF-kappaB indicates that cytoplasmic retention of NF-kappaB may be compensated for by other IkappaB proteins. Treatment of wild-type cells with tumor necrosis factor alpha (TNF-alpha) resulted in rapid, transient nuclear localization of NF-kappaB. IkappaBalpha-deficient fibroblasts are also TNF-alpha responsive, but nuclear localization of NF-kappaB is prolonged, thus demonstrating that a major irreplaceable function Of IkappaBalpha is termination of the NF-kappaB response. Consistent with these observations, and with IkappaBalpha and NF-kappaB's role in regulating inflammatory and immune responses, is the normal development Of IkappaBalpha-deficient mice. However, growth ceases 3 days after birth and death usually occurs at 7 to 10 days of age. An increased percentage of monocytes/macrophages was detected in spleen cells taken from 5-, 7-, and 9-day-old pups. Death is accompanied by severe widespread dermatitis and increased levels of TNF-alpha mRNA in the skin.
PMCID: PMC231222  PMID: 8628301
3.  I kappaB alpha physically interacts with a cytoskeleton-associated protein through its signal response domain. 
Molecular and Cellular Biology  1997;17(12):7375-7385.
The I kappaB alpha protein is a key molecular target involved in the control of NF-kappaB/Rel transcription factors during viral infection or inflammatory reactions. This NF-kappaB-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets I kappaB alpha for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the I kappaB alpha inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of I kappaB alpha as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length I kappaB alpha protein but not with N-terminal-deletion-containing versions of I kappaB alpha. I kappaB alpha interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of I kappaB alpha. Importantly, in HeLa and 293 cells, endogenous and transfected I kappaB alpha coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and I kappaB alpha colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and I kappaB alpha were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, I kappaB alpha colocalized with alpha-tubulin filaments. Taken together, these results highlight an intriguing interaction between the I kappaB alpha protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation.
PMCID: PMC232593  PMID: 9372968
4.  Characterization of a mutant cell line that does not activate NF-kappaB in response to multiple stimuli. 
Molecular and Cellular Biology  1997;17(3):1441-1449.
Numerous genes required during the immune or inflammation response as well as the adhesion process are regulated by nuclear factor kappaB (NF-kappaB). Associated with its inhibitor, I kappaB, NF-kappaB resides as an inactive form in the cytoplasm. Upon stimulation by various agents, I kappaB is proteolyzed and NF-kappaB translocates to the nucleus, where it activates its target genes. The transduction pathways that lead to I kappaB inactivation remain poorly understood. In this study, we have characterized a cellular mutant, the 70/Z3-derived 1.3E2 murine pre-B cell line, that does not activate NF-kappaB in response to several stimuli. We demonstrate that upon stimulation by lipopolysaccharide, Taxol, phorbol myristate acetate, interleukin-1, or double-stranded RNA, I kappaB alpha is not degraded, as a result of an absence of induced phosphorylation on serines 32 and 36. Neither a mutation in I kappaB alpha nor a mutation in p50 or relA, the two major subunits of NF-kappaB in this cell line, accounts for this phosphorylation defect. As well as culminating in the inducible phosphorylation of I kappaB alpha on serines 32 and 36, all the stimuli that are inactive on 1.3E2 cells exhibit a sensitivity to the antioxidant pyrrolidine dithiocarbamate (PDTC). In contrast, stimuli such as hyperosmotic shock or phosphatase inhibitors, which use PDTC-insensitive pathways, induce I kappaB alpha degradation in 1.3E2. Analysis of the redox status of 1.3E2 does not reveal any difference from wild-type 70Z/3. We also report that the human T-cell leukemia virus type 1 (HTLV-1)-derived Tax trans-activator induces NF-kappaB activity in 1.3E2, suggesting that this viral protein does not operate via the defective pathway. Finally, we show that two other I kappaB molecules, I kappaB beta and the recently identified I kappaB epsilon, are not degraded in the 1.3E2 cell line following stimulation. Our results demonstrate that 1.3E2 is a cellular transduction mutant exhibiting a defect in a step that is required by several different stimuli to activate NF-kappaB. In addition, this analysis suggests a common step in the signaling pathways that trigger I kappaB alpha, I kappaB beta, and I kappaB epsilon degradation.
PMCID: PMC231869  PMID: 9032271
5.  Docking of molecules identified in bioactive medicinal plants extracts into the p50 NF-kappaB transcription factor: correlation with inhibition of NF-kappaB/DNA interactions and inhibitory effects on IL-8 gene expression 
Background
The transcription factor NF-kappaB is a very interesting target molecule for the design on anti-tumor, anti-inflammatory and pro-apoptotic drugs. However, the application of the widely-used molecular docking computational method for the virtual screening of chemical libraries on NF-kappaB is not yet reported in literature. Docking studies on a dataset of 27 molecules from extracts of two different medicinal plants to NF-kappaB-p50 were performed with the purpose of developing a docking protocol fit for the target under study.
Results
We enhanced the simple docking procedure by means of a sort of combined target- and ligand-based drug design approach. Advantages of this combination strategy, based on a similarity parameter for the identification of weak binding chemical entities, are illustrated in this work with the discovery of a new lead compound for NF-kappaB. Further biochemical analyses based on EMSA were performed and biological effects were tested on the compound exhibiting the best docking score. All experimental analysis were in fairly good agreement with molecular modeling findings.
Conclusion
The results obtained sustain the concept that the docking performance is predictive of a biochemical activity. In this respect, this paper represents the first example of successfully individuation through molecular docking simulations of a promising lead compound for the inhibition of NF-kappaB-p50 biological activity and modulation of the expression of the NF-kB regulated IL8 gene.
doi:10.1186/1472-6807-8-38
PMCID: PMC2543017  PMID: 18768082
6.  Molecular networks discriminating mouse bladder responses to intravesical bacillus Calmette-Guerin (BCG), LPS, and TNF-α 
BMC Immunology  2008;9:4.
Background
Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression in the bladder target organ beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following chronic intravesical BCG therapy and to compare the results to non-specific pro inflammatory stimuli (LPS and TNF-α). For this purpose, C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-α. Seven days after the last instillation, the urothelium along with the submucosa was removed from detrusor muscle and the RNA was extracted from both layers for cDNA array experiments. Microarray results were normalized by a robust regression analysis and only genes with an expression above a conditional threshold of 0.001 (3SD above background) were selected for analysis. Next, genes presenting a 3-fold ratio in regard to the control group were entered in Ingenuity Pathway Analysis (IPA) for a comparative analysis in order to determine genes specifically regulated by BCG, TNF-α, and LPS. In addition, the transcriptome was precipitated with an antibody against RNA polymerase II and real-time polymerase chain reaction assay (Q-PCR) was used to confirm some of the BCG-specific transcripts.
Results
Molecular networks of treatment-specific genes generated several hypotheses regarding the mode of action of BCG. BCG-specific genes involved small GTPases and BCG-specific networks overlapped with the following canonical signaling pathways: axonal guidance, B cell receptor, aryl hydrocarbon receptor, IL-6, PPAR, Wnt/β-catenin, and cAMP. In addition, a specific detrusor network expressed a high degree of overlap with the development of the lymphatic system. Interestingly, TNF-α-specific networks overlapped with the following canonical signaling pathways: PPAR, death receptor, and apoptosis. Finally, LPS-specific networks overlapped with the LPS/IL-1 mediated inhibition of RXR. Because NF-kappaB occupied a central position in several networks, we further determined whether this transcription factor was part of the responses to BCG. Electrophoretic mobility shift assays confirmed the participation of NF-kappaB in the mouse bladder responses to BCG. In addition, BCG treatment of a human urothelial cancer cell line (J82) also increased the binding activity of NF-kappaB, as determined by precipitation of the chromatin by a NF-kappaB-p65 antibody and Q-PCR of genes bearing a NF-kappaB consensus sequence. Next, we tested the hypothesis of whether small GTPases such as LRG-47 are involved in the uptake of BCG by the bladder urothelium.
Conclusion
As expected, BCG treatment induces the transcription of genes belonging to common pro-inflammatory networks. However, BCG also induces unique genes belonging to molecular networks involved in axonal guidance and lymphatic system development within the bladder target organ. In addition, NF-kappaB seems to play a predominant role in the bladder responses to BCG therapy. Finally, in intact urothelium, BCG-GFP internalizes in LRG-47-positive vesicles.
These results provide a molecular framework for the further study of the involvement of immune and nervous systems in the bladder responses to BCG therapy.
doi:10.1186/1471-2172-9-4
PMCID: PMC2262873  PMID: 18267009
7.  Nuclear factor kappaB (NF-kappaB) pathway as a therapeutic target in rheumatoid arthritis. 
Journal of Korean Medical Science  1999;14(3):231-238.
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent joint swelling and progressive destruction of cartilage and bone. Current RA treatments are largely empirical in origin and their precise mechanism of action is uncertain. Increasing evidence shows that chronic inflammatory diseases such as RA are caused by prolonged production of proinflammatory cytokines including tumor necrosis factor (TNF) and interleukin 1 (IL-1). The nuclear factor kappaB (NF-kappaB) plays an essential role in transcriptional activation of TNF and IL-1. NF-kappaB is induced by many stimuli including TNF and IL-1, forming a positive regulatory cycle that may amplify and maintain RA disease process. NF-kappaB and enzymes involved in its activation can be a target for anti-inflammatory treatment. Aspirin and sodium salicylate inhibit activation of NF-KB by blocking IkappaB kinase, a key enzyme in NF-kappaB activation. Glucocorticoids suppress expression of inflammatory genes by binding glucocorticoid receptor with NF-kappaB, and increasing expression of inhibitory protein of NF-kappaB, IkappaBalpha. Sulfasalazine and gold compounds also inhibit NF-kappaB activation. Continuing advances in our understanding of action mechanism of antirheumatic agents will benefit the future development of RA regimens with greater efficacy and less toxicity.
PMCID: PMC3054387  PMID: 10402163
8.  Physical interactions between Ets and NF-kappaB/NFAT proteins play an important role in their cooperative activation of the human immunodeficiency virus enhancer in T cells. 
Journal of Virology  1997;71(5):3563-3573.
The transcriptional regulatory elements of many inducible T-cell genes contain adjacent or overlapping binding sites for the Ets and NF-kappaB/NFAT families of transcription factors. Similar arrays of functionally important NF-kappaB/NFAT and Ets binding sites are present in the transcriptional enhancers of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2), suggesting that this pattern of nuclear protein binding sites reflects an evolutionarily conserved mechanism for regulating inducible T-cell gene expression that has been co-opted during HIV evolution. Despite these findings, the molecular mechanisms by which Ets and NF-kappaB/NFAT proteins cooperatively regulate inducible T-cell gene expression remained unknown. In the studies described in this report, we demonstrated a physical interaction between multiple Ets and NF-kappaB/NFAT proteins both in vitro and in activated normal human T cells. This interaction is mediated by the Ets domain of Ets proteins and the C-terminal region of the Rel homology domains of NF-kappaB/NFAT proteins. In addition, the Ets-NF-kappaB/NFAT interaction requires the presence of DNA binding sites for both proteins, as it is abolished by the DNA intercalating agents propidium iodide and ethidium bromide and enhanced by the presence of synthetic oligonucleotides containing binding sites for Ets and NF-kappaB proteins. A dominant-negative mutant of NF-kappaB p50 that binds DNA but fails to interact with Ets proteins inhibits the synergistic activation of the HIV-1 and HIV-2 enhancers by NF-kappaB (p50 + p65) and Ets-1, suggesting that physical interaction between Ets and NF-kappaB proteins is required for the transcriptional activity of the HIV-1 and HIV-2 enhancers. Taken together, these findings suggest that evolutionarily conserved physical interactions between Ets and NF-kappaB/NFAT proteins are important in regulating the inducible expression of T-cell genes and viruses. These interactions represent a potential target for the development of novel immunosuppressive and antiviral therapies.
PMCID: PMC191503  PMID: 9094628
9.  Activation of transcription factor NF-kappaB by the adenovirus E3/19K protein requires its ER retention 
The Journal of Cell Biology  1996;132(4):511-522.
We have recently shown that the accumulation of diverse viral and cellular membrane proteins in the ER activates the higher eukaryotic transcription factor NF-kappaB. This defined a novel ER-nuclear signal transduction pathway, which is distinct from the previously described unfolded protein response (UPR). The well characterized UPR pathway is activated by the presence of un- or malfolded proteins in the ER. In contrast, the ER stress signal which activates the NF-kappaB pathway is not known. Here we used the adenovirus early region protein E3/19K as a model to investigate the nature of the NF-kappaB-activating signal emitted by the ER. E3/19K resides in the endoplasmic reticulum where it binds to MHC class I molecules, thereby preventing their transport to the cell surface. It is maintained in the ER by a retention signal sequence in its carboxy terminus, which causes the protein to be continuously retrieved to the ER from post-ER compartments. Mutation of this sequence allows E3/19K to reach the cell surface. We show here that expression of E3/19K potently activates a functional NF-kappaB transcription factor. The activated NF-kappaB complexes contained p50/p65 and p50/c-rel heterodimers. E3/19K interaction with MHC class I was not important for NF-kappaB activation since mutant proteins which no longer bind MHC molecules remained fully capable of inducing NF- kappaB. However, activation of both NF-kappaB DNA binding and kappaB- dependent transactivation relied on E3/19K ER retention: mutants, which were expressed on the cell surface, could no longer activate the transcription factor. This identifies the NF-kappaB-activating signal as the accumulation of proteins in the ER membrane, a condition we have termed "ER overload." We show that ER overload-mediated NF-kappaB activation but not TNF-stimulated NF-kappaB induction can be inhibited by the intracellular Ca2+ chelator TMB-8. Moreover, treatment of cells with two inhibitors of the ER-resident Ca(2+) -dependent ATPase, thapsigargin and cyclopiazonic acid, which causes a rapid release of Ca2+ from the ER, strongly activated NF-kappaB. We therefore propose that ER overload activates NF-kappaB by causing Ca2+ release from the ER. Because NF-kappaB plays a key role in mounting an immune response, ER overload caused by viral proteins may constitute a simple antiviral response with broad specificity.
PMCID: PMC2199876  PMID: 8647884
10.  Hepatitis B virus HBx protein activates transcription factor NF-kappaB by acting on multiple cytoplasmic inhibitors of rel-related proteins. 
Journal of Virology  1996;70(7):4558-4566.
The HBx protein is a small polypeptide encoded by mammalian hepadnaviruses that is essential for viral infectivity and is thought to play a role in development of hepatocellular carcinoma during chronic hepatitis B virus infection. HBx is a transactivator that stimulates Ras signal transduction pathways in the cytoplasm and certain transcription elements in the nucleus. To better understand the activities of HBx protein and its mechanism of action, we have explored the manner by which HBx activates the transcription factor NF-kappaB during transient expression. We show that HBx induces prolonged formation, in a Ras-dependent manner, of transcriptionally active NF-kappaB DNA-binding complexes, which make up the family of Rel-related proteins, p50, p52, RelA, and c-Rel. HBx was found to activate NF-kappaB through two distinct cytoplasmic pathways by acting on both the 37-kDa IkappaBalpha inhibitor and the 105-kappaDa NF-kappaB1 precursor inhibitor protein, known as p105. HBx induces phosphorylation of IkappaBalpha, a three- to fourfold reduction in IKBalpha stability, and concomitant nuclear accumulation of NF-kappaB DNA-binding complexes, similar to that reported for human T-cell leukemia virus type 1 Tax protein. In addition, HBx mediates a striking reduction in cytoplasmic p105 NF-kappaB1 inhibitor and p50 protein levels and release of RelA protein that was sequestered by the p105 inhibitor, concomitant with nuclear accumulation of NF-kappaB complexes. HBx mediated only a slight reduction in the cytoplasmic levels of NF-kappaB2 p100 protein, an additional precursor inhibitor of NF-kappaB, which is thought to be less efficiently processed or less responsive to release of NF-kappaB. No evidence was found for HBx activation of NF-kappaB by targeting acidic sphingomyelinase- controlled pathways. Studies also suggest that stimulation of NF-kappaB by HBx does not involve activation of Ras via the neutral sphingomyelin-ceramide pathway. Thus, HBx protein is shown to activate the NF-kappaB family of Rel-related proteins by acting on two distinct NF-kappaB cytoplasmic inhibitors.
PMCID: PMC190392  PMID: 8676482
11.  Rickettsia rickettsii infection of cultured human endothelial cells induces NF-kappaB activation. 
Infection and Immunity  1997;65(7):2786-2791.
Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, is an obligate intracellular bacterial organism that infects primarily the vascular endothelial cells (EC). A component of the EC response to infection is transcriptional activation, which may contribute to the thrombotic and inflammatory consequences of disease. In this study, we explore R. rickettsii-induced activation of the nuclear factor-kappaB/Rel (NF-kappaB) family of transcription factors involved in early transcriptional responses to injurious stimuli. Two NF-kappaB species were activated by infection and reacted with a double-stranded oligonucleotide probe corresponding to the kappaB binding domain of the murine kappa light-chain gene enhancer. Gel supershift analysis demonstrated the reactivity of these complexes with antibodies against p65 and p50, and the induced species were tentatively identified as p50-p50 homodimers and p50-p65 heterodimers. Semiquantitative reverse transcription-PCR analysis revealed dramatic increases in the steady-state levels of mRNA coding for the inhibitory subunit of NF-kappaB (IkappaB alpha), transcription of which is enhanced by the binding of NF-kappaB within the IkappaB alpha promoter region. NF-kappaB activation was first detected 1.5 h following infection and was biphasic, with an early peak of activation at approximately 3 h, a return to baseline levels at 14 h, and even higher levels of activation at 24 h. It is likely that NF-kappaB activation requires cellular uptake of R. rickettsii, since treatment of EC with cytochalasin B during infection to block entry inhibited activation by only 70% at 3 h. R. rickettsii-induced activation of NF-kappaB may be an important controlling factor in the transcriptional responses of EC to infection with this obligate intracellular organism.
PMCID: PMC175393  PMID: 9199451
12.  TNF Superfamily: A Growing Saga of Kidney Injury Modulators 
Mediators of Inflammation  2010;2010:182958.
Members of the TNF superfamily participate in kidney disease. Tumor necrosis factor (TNF) and Fas ligand regulate renal cell survival and inflammation, and therapeutic targeting improves the outcome of experimental renal injury. TNF-related apoptosis-inducing ligand (TRAIL and its potential decoy receptor osteoprotegerin are the two most upregulated death-related genes in human diabetic nephropathy. TRAIL activates NF-kappaB in tubular cells and promotes apoptosis in tubular cells and podocytes, especially in a high-glucose environment. By contrast, osteoprotegerin plays a protective role against TRAIL-induced apoptosis. Another family member, TNF-like weak inducer of apoptosis (TWEAK induces inflammation and tubular cell death or proliferation, depending on the microenvironment. While TNF only activates canonical NF-kappaB signaling, TWEAK promotes both canonical and noncanonical NF-kappaB activation in tubular cells, regulating different inflammatory responses. TWEAK promotes the secretion of MCP-1 and RANTES through NF-kappaB RelA-containing complexes and upregulates CCl21 and CCL19 expression through NF-kappaB inducing kinase (NIK-) dependent RelB/NF-kappaB2 complexes. In vivo TWEAK promotes postnephrectomy compensatory renal cell proliferation in a noninflammatory milieu. However, in the inflammatory milieu of acute kidney injury, TWEAK promotes tubular cell death and inflammation. Therapeutic targeting of TNF superfamily cytokines, including multipronged approaches targeting several cytokines should be further explored.
doi:10.1155/2010/182958
PMCID: PMC2952810  PMID: 20953353
13.  Loss of NF-kappaB activity during cerebral ischemia and TNF cytotoxicity. 
Molecular Medicine  1999;5(6):372-381.
Recent evidence implicates tumor necrosis factor (TNF), a cytokine with both cytotoxic and cytoprotective activities, in the pathogenesis of cerebral ischemia. The development of TNF cytotoxicity is dependent upon the balance between the activities of intracellular signaling pathways that mediate either apoptotic or anti-apoptotic effects. One critical protective signaling mechanism is the activation of nuclear factor (NF)-kappaB, a ubiquitous transcription factor that regulates expression of anti-apoptotic gene products. Here we show the distribution and kinetics of NF-kappaB activation and the correlation between loss of NF-kappaB activity, TNF up-regulation, and apoptosis in a standardized rat model of focal cerebral ischemia. We observed a rapid and progressive ischemia-induced loss of p65 immunoreactivity within the ischemic core and nearby penumbra. These findings were confirmed by Western blot analysis of nuclear extracts and by electrophoretic mobility shift assay. The anatomical area of suppressed NF-kappaB activity overlapped significantly with the zones of TNF overexpression and apoptosis. Loss of NF-kappaB activity and increased TNF expression preceded the onset of cell death. Direct evidence that loss of NF-kappaB activity can sensitize brain cells to TNF cytotoxicity was obtained in vitro by co-administration of MG-132, an inhibitor of NF-kappaB activation, and TNF to neuronal-like and glial-like cell cultures. Inhibition of NF-kappaB significantly increased the sensitivity of these cultures to TNF cytotoxicity, indicating that the observed loss of neuronal NF-kappaB activity during cerebral ischemia can participate in the development of TNF-induced cytotoxicity.
Images
PMCID: PMC2230435  PMID: 10415162
14.  Activation of NF-kappaB by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells. 
Journal of Clinical Investigation  1998;101(11):2598-2605.
PMN-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease. Epithelial cells respond to organisms such as Pseudomonas aeruginosa, the major pathogen in CF, by expressing the leukocyte chemokine IL-8. Experiments were performed using several different types of respiratory epithelial cells that demonstrate that ligation of ceramide-associated receptors on epithelial surfaces by P. aeruginosa pili is a major stimulus for the translocation of transcription factor nuclear factor (NF)-kappaB and initiation of IL-8 expression by epithelial cells. Using electrophoretic mobility shift assays and Western hybridizations, nuclear NF-kappaB was found shortly after epithelial cells were stimulated by either whole organisms, isolated pili, or antibody to the pilin receptor asialoGM1. IB3 cells, which express mutations in cystic fibrosis transmembrane conductance regulator (CFTR) (DeltaF508/W1282X), were noted to have significantly greater amounts of endogenous nuclear NF-kappaB, but not the transcription factor C/EBP, than CF cells corrected by episomal copies of normal CFTR (C-38) or IB3 cells grown at a permissive temperature (25 degreesC). Activation of NF-kappaB and subsequent IL-8 expression in epithelial cells can result from activation of at least two pathways: an exogenous signaling cascade that is activated by ligation of ceramide-associated adhesins such as P. aeruginosa pilin, or endogenous stimulation, suggested to be a consequence of cell stress caused by the accumulation of mutant CFTR in the endoplasmic reticulum.
PMCID: PMC508849  PMID: 9616231
15.  NF kappaB expression increases and CFTR and MUC1 expression decreases in the endometrium of infertile patients with hydrosalpinx: a comparative study 
Background
Hydrosalpinx are associated with infertility, due to reduced rates of implantation and increased abortion rates. The aims of this study were to investigate the expression of cystic fibrosis transmembrane conductance regulator (CFTR), nuclear factor kappa B (NF KappaB) and mucin-1 (MUC-1), and analyze the correlation between the expression of CFTR and NF KappaB or MUC1, in the endometrium of infertile women with and without hydrosalpinx.
Methods
Thirty-one infertile women with laparoscopy-confirmed unilateral or bilateral hydrosalpinx and 20 infertile women without hydrosalpinx or pelvic inflammatory disease (control group) were recruited. Endometrial biopsy samples were collected and the expression of CFTR, NF KappaB and MUC1 were analyzed using immunohistochemistry and quantitative real-time PCR.
Results
CFTR, NF KappaB and MUC1 mRNA and protein expression tended to increase in the secretory phase compared to the proliferative phase in both groups; however, these differences were not significantly different. The endometrium of infertile patients with hydrosalpinx had significantly higher NF KappaB mRNA and protein expression, and significantly lower CFTR and MUC1 mRNA and protein expression, compared to control infertile patients. A positive correlation was observed between CFTR and MUC1 mRNA expression (r = 0.65, P < 0.05); a negative correlation was observed between CFTR mRNA and NF KappaB mRNA expression (r = −0.59, P < 0.05).
Conclusions
Increased NF KappaB expression and decreased CFTR and MUC1 expression in the endometrium of infertile patients with hydrosalpinx reinforce the involvement of a molecular mechanism in the regulation of endometrial receptivity.
doi:10.1186/1477-7827-10-86
PMCID: PMC3551815  PMID: 23061681
Cystic fibrosis transmembrane conductance regulator; Endometrium; Hydrosalpinx; Mucin-1; Nuclear factor kappa B
16.  microRNA-146a inhibits G protein-coupled receptor-mediated activation of NF-κB by targeting CARD10 and COPS8 in gastric cancer 
Molecular Cancer  2012;11:71.
Background
Gastric cancer is the second most common cause of cancer-related death in the world. Inflammatory signals originating from gastric cancer cells are important for recruiting inflammatory cells and regulation of metastasis of gastric cancer. Several microRNAs (miRNA) have been shown to be involved in development and progression of gastric cancer. miRNA-146a (miR-146a) is a modulator of inflammatory signals, but little is known about its importance in gastric cancer. We therefore wanted to identify targets of miR-146a in gastric cancer and examine its biological roles.
Results
The expression of miR-146a was evaluated by quantitative PCR (qPCR) and found up-regulated in the gastrin knockout mice, a mouse model of gastric cancer, and in 73% of investigated human gastric adenocarcinomas. Expression of miR-146a by gastric cancer cells was confirmed by in situ hybridization. Global analysis of changes in mRNA levels after miR-146a transfection identified two transcripts, caspase recruitment domain-containing protein 10 (CARD10) and COP9 signalosome complex subunit 8 (COPS8), as new miR-146a targets. qPCR, Western blotting and luciferase assays confirmed these transcripts as direct miR-146a targets. CARD10 and COPS8 were shown to be part of the G protein-coupled receptor (GPCR) pathway of nuclear factor-kappaB (NF-kappaB) activation. Lysophosphatidic acid (LPA) induces NF-kappaB activation via this pathway and over-expression of miR-146a inhibited LPA-induced NF-kappaB activation, reduced LPA-induced expression of tumor-promoting cytokines and growth factors and inhibited monocyte attraction.
Conclusions
miR-146a expression is up-regulated in a majority of gastric cancers where it targets CARD10 and COPS8, inhibiting GPCR-mediated activation of NF-kappaB, thus reducing expression of NF-kappaB-regulated tumor-promoting cytokines and growth factors. By targeting components of several NF-kappaB-activating pathways, miR-146a is a key component in the regulation of NF-kappaB activity.
doi:10.1186/1476-4598-11-71
PMCID: PMC3515505  PMID: 22992343
Stomach cancer; Non-coding RNA; Cytokines
17.  Sulfasalazine: a potent and specific inhibitor of nuclear factor kappa B. 
Journal of Clinical Investigation  1998;101(5):1163-1174.
Transcription factors of the NF-kappaB/Rel family are critical for inducible expression of multiple genes involved in inflammatory responses. Sulfasalazine and its salicylate moiety 5-aminosalicylic acid are among the most effective agents for treating inflammatory bowel disease and rheumatoid arthritis. However, the mode of action of these drugs remains unclear. Here we provide evidence that the transcription factor NF-kappaB is a target of sulfasalazine-mediated immunosuppression. Treatment of SW620 colon cells with sulfasalazine inhibited TNFalpha-, LPS-, or phorbol ester- induced NF-kappaB activation. NF-kappaB-dependent transcription was inhibited by sulfasalazine at micro- to millimolar concentrations. In contrast, 5-aminosalicylic acid or sulfapyridine did not block NF-kappaB activation at all doses tested. TNFalpha-induced nuclear translocation of NF-kappaB was prevented by sulfasalazine through inhibition of IkappaBalpha degradation. When blocking proteasome-mediated degradation of IkappaBalpha, we could demonstrate that sulfasalazine interfered with IkappaBalpha phosphorylation, suggesting a direct effect on an IkappaBalpha kinase or on an upstream signal. Inhibition of NF-kappaB activation seems to be specific since other DNA-binding activities such as AP1 were not affected. These results demonstrate that sulfasalazine is a potent and specific inhibitor of NF-kappaB activation, and thus may explain some of the known biological properties of sulfasalazine.
PMCID: PMC508669  PMID: 9486988
18.  PDP-1 Links the TGF-β and IIS Pathways to Regulate Longevity, Development, and Metabolism 
PLoS Genetics  2011;7(4):e1001377.
The insulin/IGF-1 signaling (IIS) pathway is a conserved regulator of longevity, development, and metabolism. In Caenorhabditis elegans IIS involves activation of DAF-2 (insulin/IGF-1 receptor tyrosine kinase), AGE-1 (PI 3-kinase), and additional downstream serine/threonine kinases that ultimately phosphorylate and negatively regulate the single FOXO transcription factor homolog DAF-16. Phosphatases help to maintain cellular signaling homeostasis by counterbalancing kinase activity. However, few phosphatases have been identified that negatively regulate the IIS pathway. Here we identify and characterize pdp-1 as a novel negative modulator of the IIS pathway. We show that PDP-1 regulates multiple outputs of IIS such as longevity, fat storage, and dauer diapause. In addition, PDP-1 promotes DAF-16 nuclear localization and transcriptional activity. Interestingly, genetic epistasis analyses place PDP-1 in the DAF-7/TGF-β signaling pathway, at the level of the R-SMAD proteins DAF-14 and DAF-8. Further investigation into how a component of TGF-β signaling affects multiple outputs of IIS/DAF-16, revealed extensive crosstalk between these two well-conserved signaling pathways. We find that PDP-1 modulates the expression of several insulin genes that are likely to feed into the IIS pathway to regulate DAF-16 activity. Importantly, dysregulation of IIS and TGF-β signaling has been implicated in diseases such as Type 2 Diabetes, obesity, and cancer. Our results may provide a new perspective in understanding of the regulation of these pathways under normal conditions and in the context of disease.
Author Summary
Cells in the body respond to a variety of on/off signals that are relayed in a defined spatial and temporal manner. These signals influence several processes such as growth, fat storage, and the repair of damaged molecules. As humans age, the onset of diseases such as Type 2 Diabetes, obesity, and cancer often results from an imbalance in the levels of on/off signals in the cell. The insulin/IGF-1 signaling pathway is an important regulator of longevity, development, and metabolism across phylogeny. While the protein kinases that activate this pathway have been well studied, less is known about the protein phosphatases that tune down the signals. The roundworm C. elegans has been an excellent model system to study the role of insulin/IGF-1 signaling in the aging process. Here, we identify a new phosphatase that negatively regulates the insulin/IGF-1 pathway to enhance longevity and stress-resistance. Interestingly, the phosphatase achieves this function by tuning down the activity of a conserved TGF-β pathway, a pathway important for development. By reducing TGF-β pathway activity, this phosphatase decreases expression of insulin molecules that may stimulate the insulin/IGF-1 pathway. Our studies not only unravel a new regulator of these pathways, but also point to how they are more linked than previously thought. Both insulin/IGF-1 and TGF-β signaling have been implicated in age-associated diseases, and understanding their connection will provide us with potential therapeutic avenues.
doi:10.1371/journal.pgen.1001377
PMCID: PMC3080858  PMID: 21533078
19.  Bmi-1 promotes the aggressiveness of glioma via activating the NF-kappaB/MMP-9 signaling pathway 
BMC Cancer  2012;12:406.
Background
The prognosis of human glioma is poor, and the highly invasive nature of the disease represents a major impediment to current therapeutic modalities. The oncoprotein B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) has been linked to the development and progression of glioma; however, the biological role of Bmi-1 in the invasion of glioma remains unclear.
Methods
A172 and LN229 glioma cells were engineered to overexpress Bmi-1 via stable transfection or to be silenced for Bmi-1 expression using RNA interfering method. Migration and invasiveness of the engineered cells were assessed using wound healing assay, Transwell migration assay, Transwell matrix penetration assay and 3-D spheroid invasion assay. MMP-9 expression and activity were measured using real-time PCR, ELISA and the gelatin zymography methods. Expression of NF-kappaB target genes was quantified using real-time PCR. NF-kappaB transcriptional activity was assessed using an NF-kappaB luciferase reporter system. Expression of Bmi-1 and MMP-9 in clinical specimens was analyzed using immunohistochemical assay.
Results
Ectopic overexpression of Bmi-1 dramatically increased, whereas knockdown of endogenous Bmi-1 reduced, the invasiveness and migration of glioma cells. NF-kappaB transcriptional activity and MMP-9 expression and activity were significantly increased in Bmi-1-overexpressing but reduced in Bmi-1-silenced cells. The reporter luciferase activity driven by MMP-9 promoter in Bmi-1-overexpressing cells was dependent on the presence of a functional NF-kappaB binding site, and blockade of NF-kappaB signaling inhibited the upregulation of MMP-9 in Bmi-1 overexpressing cells. Furthermore, expression of Bmi-1 correlated with NF-kappaB nuclear translocation as well as MMP-9 expression in clinical glioma samples.
Conclusions
Bmi-1 may play an important role in the development of aggressive phenotype of glioma via activating the NF-kappaB/MMP-9 pathway and therefore might represent a novel therapeutic target for glioma.
doi:10.1186/1471-2407-12-406
PMCID: PMC3502583  PMID: 22967049
Bmi-1; Glioma; Invasion; MMP-9; NF-kappaB
20.  Signaling from toxic metals to NF-kappaB and beyond: not just a matter of reactive oxygen species. 
Environmental Health Perspectives  2002;110(Suppl 5):807-811.
The nuclear factor kappa B (NF-kappaB) family of transcription factors controls expression of a number of early response genes associated with inflammatory responses, cell growth, cell cycle progression, and neoplastic transformation. These genes include a multitude of cytokines, chemokines, adhesion molecules, immune receptors, stress proteins, apoptotic or anti-apoptotic regulators, and several oncogenes. Accumulating evidence indicates that a variety of toxic metals are able to affect the activation or activity of NF-kappaB, but the molecular mechanisms involved in this process remain largely unknown. The signaling pathways mediating cytokine- or microorganism-induced NF-kappaB activation have been well established recently. Whether the same signaling systems are involved in metal-induced NF-kappaB activation, however, is unclear. In the present review, we have attempted to evaluate and update the possible mechanisms of metal signals on the activation and function of NF-kappaB.
PMCID: PMC1241250  PMID: 12426136
21.  Distinct functional properties of IkappaB alpha and IkappaB beta. 
Molecular and Cellular Biology  1997;17(9):5386-5399.
The biological activity of the transcription factor NF-kappaB is controlled mainly by the IkappaB alpha and IkappaB beta proteins, which restrict NF-kappaB to the cytoplasm and inhibit its DNA binding activity. Here, we carried out experiments to determine and compare the mechanisms by which IkappaB alpha and IkappaB beta inhibit NF-kappaB-dependent transcriptional activation. First, we found that in vivo IkappaB alpha is a stronger inhibitor of NF-kappaB than is IkappaB beta. This difference is directly correlated with their abilities to inhibit NF-kappaB binding to DNA in vitro and in vivo. Moreover, IkappaB alpha, but not IkappaB beta, can remove NF-kappaB from functional preinitiation complexes in in vitro transcription experiments. Second, we showed that both IkappaBs function in vivo not only in the cytoplasm but also in the nucleus, where they inhibit NF-kappaB binding to DNA. Third, the inhibitory activity of IkappaB beta, but not that of IkappaB alpha, is facilitated by phosphorylation of the C-terminal PEST sequence by casein kinase II and/or by the interaction of NF-kappaB with high-mobility group protein I (HMG I) on selected promoters. The unphosphorylated form of IkappaB beta forms stable ternary complexes with NF-kappaB on the DNA either in vitro or in vivo. These experiments suggest that IkappaB alpha works as a postinduction repressor of NF-kappaB independently of HMG I, whereas IkappaB beta functions preferentially in promoters regulated by the NF-kappaB/HMG I complexes.
PMCID: PMC232389  PMID: 9271416
22.  Modulation of the Expression of the Proinflammatory IL-8 Gene in Cystic Fibrosis Cells by Extracts Deriving from Olive Mill Waste Water 
A persistent recruitment of neutrophils in the bronchi of cystic fibrosis (CF) patients contributes to aggravate the airway tissue damage, suggesting the importance of modulating the expression of chemokines, including IL-8 during the management of the CF patients. Polyphenols rich extracts derived from waste water from olive mill, obtained by a molecular imprinting approach, have been investigated in order to discover compounds able to reduce IL-8 expression in human bronchial epithelial cells (IB3-1 cells), derived from a CF patient with a ΔF508/W1282X mutant genotype and stimulated with TNF-alpha. Initially, electrophoretic mobility shift assays (EMSAs) were performed to determine whether the different active principles were able to inhibit the binding between transcription factor (TF) NF-kappaB and DNA consensus sequences. Among different representative active principles present in the extract, three compounds were selected, apigenin, oleuropein, and cyanidin chloride, which displayed remarkable activity in inhibiting NF-kappaB/DNA complexes. Utilizing TNF-alpha-treated IB3-1 cells as experimental model system, we demonstrated that apigenin and cyanidin chloride are able to modulate the expression of the NF-kappaB-regulated IL-8 gene, while oleuropein showed no effect in regulating the expression of the gene IL-8.
doi:10.1155/2013/960603
PMCID: PMC3723063  PMID: 23935691
23.  An isoform-specific mutant reveals a role of PDP1ε in the circadian oscillator 
The Drosophila PAR domain protein 1 (Pdp1) gene encodes a transcription factor with multiple functions. One isoform, PDP1ε, was proposed to be an essential activator of the core clock gene, Clock (Clk). However, a central clock function for PDP1ε was recently disputed, and genetic analysis has been difficult due to developmental lethality of Pdp1 null mutants. Here we report the discovery of a mutation that specifically disrupts the Pdp1ε isoform. Homozygous Pdp1ε mutants are viable and exhibit arrhythmic circadian behavior in constant darkness and also in the presence of light:dark cycles. Importantly, the mutants show diminished expression of CLK and PERIOD (PER) in the central clock cells. In addition, expression of Pigment Dispersing Factor (PDF) is reduced in a subset of the central clock cells. Loss of Pdp1ε also alters the phosphorylation status of the CLK protein and disrupts cyclic expression of a per-luciferase reporter in peripheral clocks under free-running conditions. Transgenic expression of PDP1ε in clock neurons of Pdp1ε mutants can restore rhythmic circadian behavior. However, transgenic expression of CLK in these mutants rescues the expression of PER in the central clock, but fails to restore behavioral rhythms, suggesting that PDP1ε has effects outside the core molecular clock. Taken together, these data support a model in which PDP1ε functions in the central circadian oscillator as well as in the output pathway.
doi:10.1523/JNEUROSCI.2133-09.2009
PMCID: PMC2757269  PMID: 19726650
Pdp1ε; Clock; period; pigment dispersing factor (PDF); circadian rhythms; Drosophila
24.  The B1-agonist [des-Arg10]-kallidin activates transcription factor NF-kappaB and induces homologous upregulation of the bradykinin B1-receptor in cultured human lung fibroblasts. 
Journal of Clinical Investigation  1998;101(10):2080-2091.
The bradykinin B1-receptor is strongly upregulated under chronic inflammatory conditions. However, the mechanism and reason are not known. Because a better understanding of the mechanism of the upregulation will help in understanding its potential importance in inflammation, we have studied the molecular mechanism of B1-receptor upregulation in cultured human lung fibroblasts (IMR 90) in response to IL-1beta and the B1-agonist [des-Arg10]-kallidin. We show that treatment of human IMR 90 cells by IL-1beta stimulates the expression of both B1-receptor mRNA and protein. The latter was studied by Western blot analysis using antipeptide antibodies directed against the COOH-terminal part of the human B1-receptor. We furthermore report the novel observation that the B1-receptor is upregulated by its own agonist which was completely blocked by the specific B1-antagonist [des-Arg10-Leu9]-kallidin, indicating an upregulation entirely mediated through cell surface B1-receptors. The increased population of B1-receptors was functionally coupled as exemplified by an enhancement of the B1-agonist induced increase in free cytosolic calcium. Upregulation by the B1-agonist was blocked by a specific protein kinase C inhibitor. B1-agonist-induced upregulation was correlated to the induction of transcription factor nuclear factor kappaB (NF-kappaB) which efficiently bound to the NF-kappaB-like sequence located in the promoter region of the human B1-receptor gene. This correlation was further confirmed by reporter gene assays which showed that this NF-kappaB-like sequence, in the B1-receptor promoter context, could contribute to IL-1beta and DLBK-induced B1-receptor transcription activation, and by the effect of NF-kappaB inhibitor pyrrolidinedithiocarbamate which diminished both B1-receptor upregulation and NF-kappaB activation. NF-kappaB is now recognized as a key inflammatory mediator which is activated by the B1-agonist but which is also involved in B1-receptor upregulation.
PMCID: PMC508796  PMID: 9593764
25.  Regulation of Mcl-1 by constitutive activation of NF-kappaB contributes to cell viability in human esophageal squamous cell carcinoma cells 
BMC Cancer  2014;14:98.
Background
Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies with a 5-year survival rate less than 15%. Understanding of the molecular mechanisms involved in the pathogenesis of ESCC becomes critical to develop more effective treatments.
Methods
Mcl-1 expression was measured by reverse transcription (RT)-PCR and Western blotting. Human Mcl-1 promoter activity was evaluated by reporter gene assay. The interactions between DNA and transcription factors were confirmed by electrophoretic mobility shift assay (EMSA) in vitro and by chromatin immunoprecipitation (ChIP) assay in cells.
Results
Four human ESCC cell lines, TE-1, Eca109, KYSE150 and KYSE510, are revealed increased levels of Mcl-1 mRNA and protein compare with HaCaT, an immortal non-tumorigenic cell line. Results of reporter gene assays demonstrate that human Mcl-1 promoter activity is decreased by mutation of kappaB binding site, specific NF-kappaB inhibitor Bay11-7082 or dominant inhibitory molecule DNMIkappaBalpha in TE-1 and KYSE150 cell lines. Mcl-1 protein level is also attenuated by Bay11-7082 treatment or co-transfection of DNMIkappaBalpha in TE-1 and KYSE150 cells. EMSA results indicate that NF-kappaB subunits p50 and p65 bind to human Mcl-1-kappaB probe in vitro. ChIP assay further confirm p50 and p65 directly bind to human Mcl-1 promoter in intact cells, by which regulates Mcl-1 expression and contributes to the viability of TE-1 cells.
Conclusions
Our data provided evidence that one of the mechanisms of Mcl-1 expression in human ESCC is regulated by the activation of NF-kappaB signaling. The newly identified mechanism might provide a scientific basis for developing effective approaches to treatment human ESCC.
doi:10.1186/1471-2407-14-98
PMCID: PMC3930545  PMID: 24529193
Esophageal squamous cell carcinoma; Gene regulation; NF-κB; Mcl-1; Cell viability

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