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1.  Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth. 
Molecular and Cellular Biology  1997;17(7):3629-3639.
Breast cancers often progress from a hormone-dependent, nonmetastatic, antiestrogen-sensitive phenotype to a hormone-independent, antiestrogen- and chemotherapy-resistant phenotype with highly invasive and metastatic growth properties. This progression is usually accompanied by altered function of the estrogen receptor (ER) or outgrowth of ER-negative cancer cells. To understand the molecular mechanisms responsible for metastatic growth of ER-negative breast cancers, the activities of the transcription factor NF-kappaB (which modulates the expression of genes involved in cell proliferation, differentiation, apoptosis, and metastasis) were compared in ER-positive (MCF-7 and T47-D) and ER-negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines. NF-kappaB, which is usually maintained in an inactive state by protein-protein interaction with inhibitor IkappaBs, was found to be constitutively active in ER-negative breast cancer cell lines. Constitutive DNA binding of NF-kappaB was also observed with extracts from ER-negative, poorly differentiated primary breast tumors. Progression of the rat mammary carcinoma cell line RM22-F5 from an ER-positive, nonmalignant phenotype (E phenotype) to an ER-negative, malignant phenotype (F phenotype) was also accompanied by constitutive activation of NF-kappaB. Analysis of individual subunits of NF-kappaB revealed that all ER-negative cell lines, including RM22-F5 cells of F phenotype, contain a unique 37-kDa protein which is antigenically related to the RelA subunit. Cell-type-specific differences in IkappaB alpha, -beta, and -gamma were also observed. In transient-transfection experiments, constitutive activity of an NF-kappaB-dependent promoter was observed in MDA-MB-231 and RM22-F5 cells of F phenotype, and this activity was efficiently repressed by cotransfected ER. Since ER inhibits the constitutive as well as inducible activation function of NF-kappaB in a dose-dependent manner, we propose that breast cancers that lack functional ER overexpress NF-kappaB-regulated genes. Furthermore, since recent data indicate that NF-kappaB protects cells from tumor necrosis factor alpha-, ionizing radiation-, and chemotherapeutic agent daunorubicin-mediated apoptosis, our results provide an explanation for chemotherapeutic resistance in ER-negative breast cancers.
PMCID: PMC232215  PMID: 9199297
2.  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.
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.
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.
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.
PMCID: PMC3515505  PMID: 22992343
Stomach cancer; Non-coding RNA; Cytokines
3.  Dopamine stimulates expression of the human immunodeficiency virus type 1 via NF-kappaB in cells of the immune system. 
Nucleic Acids Research  1999;27(16):3291-3299.
Recent studies have reported that lymphocytes produce, transport and bind dopamine present in plasma. However, the action of dopamine on HIV-1 gene expression in cells of the immune system has not yet been examined. Here, we have investigated the regulation of HIV-1 expression by dopamine in Jurkat T cells and in primary blood mononuclear cells (PBMC). HIV-1 replication was increased by dopamine, which correlated with the increased levels of HIV-1 transactivation. Our transient expression data revealed that dopamine stimulated transcription through the NF-kappaB element present in the long terminal repeat. The importance of NF-kappaB sites was confirmed by using vectors containing wild-type or mutant kappaB sites in a heterologous promoter. Consistent with the role of NF-kappaB in mediating dopamine responsiveness, the proteasome inhibitor MG132 abolished dopamine-induced transcriptional activation. We further explored the effect of dopamine in the presence of phorbol esters or tumor necrosis factor-alpha (TNF-alpha) known to activate NF-kappaB. The combination of dopamine and TNF-alpha led to a stimulation of HIV-1 transcription and replication. However, in contrast with TNF-alpha, dopamine treatment did not affect NF-kappaB DNA binding activity nor the concentrations of p50, p65 and IkappaB-alpha proteins, which suggests a distinct NF-kappaB activation mechanism. These results reveal a new link between the dopamine system, cytokine signaling pathway and regulation of gene expression via the involvement of NF-kappaB in T cells and PBMC.
PMCID: PMC148562  PMID: 10454636
4.  The kappaB sites in the human immunodeficiency virus type 1 long terminal repeat enhance virus replication yet are not absolutely required for viral growth. 
Journal of Virology  1997;71(7):5495-5504.
The dependence of human immunodeficiency virus type 1 (HIV-1) on its NF-kappaB binding sites (kappaB sites) for replication in transformed and primary T-cell targets was examined by infecting cells with HIV-1 reporter viruses containing kappaB site enhancer mutations. Viral transcription was measured either with luciferase-expressing HIV-1 that infects for a single round or by flow cytometric analyses with HIV-1 expressing placental alkaline phosphatase (PLAP) or green-fluorescent protein (GFP). Both PLAP- and GFP-expressing viruses spread from cell to cell and allowed analysis of viral gene expression patterns in single cells. Infection of a panel of T-cell lines with different basal levels of NF-kappaB demonstrated a direct correlation between the amount of constitutive nuclear NF-kappaB and the degree to which a wild-type virus outperformed kappaB site mutants. One T-cell line with a constitutively high level of nuclear NF-kappaB, PM1, showed a 20-fold decrease in transcription when its kappaB sites were mutated. In contrast, in a T-cell line with a low basal level of NF-kappaB, SupT1, mutation of the kappaB site in the enhancer had no effect on viral transcription or growth rate. Phytohemagglutinin-activated peripheral blood mononuclear cells showed a large dependence on the kappaB sites for optimal virus growth. Viruses without marker genes corroborated the finding that mutations to the kappaB sites impair virus production in cells with a high basal level of NF-kappaB. These data show that in T cells, HIV-1 can use NF-kappaB to enhance its growth but the virus is clearly able to grow in its absence.
PMCID: PMC191791  PMID: 9188623
5.  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
6.  Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer. 
Journal of Clinical Investigation  1997;100(12):2952-2960.
Expression of nuclear factor-kappaB (NF-kappaB)/Rel transcription factors has recently been found to promote cell survival, inhibiting the induction of apoptosis. In most cells other than B lymphocytes, NF-kappaB/Rel is inactive, sequestered in the cytoplasm. For example, nuclear extracts from two human untransformed breast epithelial cell lines expressed only very low levels of NF-kappaB. Unexpectedly, nuclear extracts from two human breast tumor cell lines displayed significant levels of NF-kappaB/Rel. Direct inhibition of this NF-kappaB/ Rel activity in breast cancer cells induced apoptosis. High levels of NF-kappaB/Rel binding were also observed in carcinogen-induced primary rat mammary tumors, whereas only expectedly low levels were seen in normal rat mammary glands. Furthermore, multiple human breast cancer specimens contained significant levels of nuclear NF-kappaB/Rel subunits. Thus, aberrant nuclear expression of NF-kappaB/Rel is associated with breast cancer. Given the role of NF-kappaB/Rel factors in cell survival, this aberrant activity may play a role in tumor progression, and represents a possible therapeutic target in the treatment of these tumors.
PMCID: PMC508506  PMID: 9399940
7.  Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability. 
Molecular and Cellular Biology  1996;16(4):1401-1409.
The NF-kappaB/Rel transcription factors participate in the activation of immune system regulatory genes and viral early genes including the human immunodeficiency virus type 1 long terminal repeat. NF-kappaB/Rel proteins are coupled to inhibitory molecules, collectively termed IkappaB, which are responsible for cytoplasmic retention of NF-kappaB. Cell activation leads to the phosphorylation and degradation of IkappaBalpha, permitting NG-kappaB/Rel translocation to the nucleus and target gene activation. To further characterize the signaling events that contribute to IkappaBalpha phosphorylation, a kinase activity was isolated from Jurkat T cells that specifically interacted with IkappaBalpha in an affinity chromatography step and phosphorylated IkappaBalpha with high specificity in vitro. By using an in-gel kinase assay with recombinant IkappaBalpha as substrate, two forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria and immunological cross-reactivity identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Deletion mutants of IkappaBalpha delta1 to delta4) localized phosphorylation to the C-terminal PEST domain of IkappaBalpha. Point mutation of residues T-291, S-283, and T-299 dramatically reduced phosphorylation of IkappaBalpha by the kinase in vitro. NIH-3T3 cells that stably expressed wild-type IkappaBalpha (wtIkappaB), double-point-mutated IkappaBalpha (T291A, S283A), or triple-point-mutated IkappaBalpha (T291A, S283A, T299A) under the control of the tetracycline-responsive promoter were generated. Constitutive phosphorylation of the triple point mutant was eliminated in vivo, although tumor necrosis factor-inducible IkappaBalpha degradation was unaffected. In cell lines and in transiently transfected cells, mutation of the CKII sites in IkappaBalpha resulted in a protein with increased intrinsic stability. Together with results demonstrating a role for N-terminal sites in inducer-mediated phosphorylation and degradation of IkappaBalpha, these studies indicate that CKII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of IkappaBalpha.
PMCID: PMC231124  PMID: 8657113
8.  miR-221/222 Targets Adiponectin Receptor 1 to Promote the Epithelial-to-Mesenchymal Transition in Breast Cancer 
PLoS ONE  2013;8(6):e66502.
The epithelial-to-mesenchymal transition (EMT) is a highly conserved physiological program involved in development and tissue repair; however, its aberrant activation has been implicated in accelerating the progression of a variety of cancers. In breast cancer, the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) are differentially expressed in the clinically more aggressive basal-like subtype compared to luminal subtype of breast cancer and upregulation of miR-221/222 induces the EMT by targeting the 3′ untranslated region (3′UTR) of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1). The complete mechanism through which miR-221/222 promotes the EMT, however, is not fully understood. We identified adiponectin receptor 1 (ADIPOR1), a receptor for the adipocytokine adiponectin, as a direct target of miR-221/222. ADIPOR1 is expressed at higher levels in the luminal compared to the basal-like subtype of breast cancer cell lines, which can be reduced by miR-221/222 targeting of its 3’UTR. In addition, miR-221/222 were negatively correlated with ADIPOR1 expression across breast cancer cell lines and tumors. ADIPOR1 depletion by siRNA in MCF10A cells induced the EMT and increased cell invasion. Depletion of ADIPOR1 by siRNA induced activation of the canonical nuclear factor-kappaB (NF-κB) and subsequent phosphorylation of signal transducer and activator of transcription 3 (STAT3) in an interleukin 6 (IL6)-dependent manner. Finally, overexpression of ADIPOR1 in the basal-like cell line, MDA-MB-231, attenuated cell invasion and promoted the mesenchymal-to-epithelial transition (MET). We conclude that ADIPOR1 negatively regulates EMT in breast cancer and provides an additional node by which miR-221/222 induces the EMT. These results suggest that ADIPOR1 may play an important role in breast cancer progression and metastasis, and could potentially offer an alternative therapeutic strategy for basal-like breast cancer patients.
PMCID: PMC3679042  PMID: 23776679
9.  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
10.  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
11.  A new member of the I kappaB protein family, I kappaB epsilon, inhibits RelA (p65)-mediated NF-kappaB transcription. 
Molecular and Cellular Biology  1997;17(10):6184-6190.
A novel member of the I kappaB family has been identified as a protein that associated with the p50 subunit of NF-kappaB in a yeast two-hybrid screen. Similar to previously known I kappaB proteins, this member, I kappaB epsilon, has six consecutive ankyrin repeats. I kappaB epsilon mRNA is widely expressed in different human tissues, with highest levels in spleen, testis, and lung. I kappaB epsilon interacts with different NF-kappaB proteins, including p65 (RelA), c-Rel, p50, and p52, in vitro and in vivo and inhibits the DNA-binding activity of both p50-p65 and p50-c-Rel complexes effectively. Endogenous and transfected NF-kappaB (RelA-dependent) transcriptional activation is inhibited by I kappaB epsilon. I kappaB epsilon mRNA is expressed at different levels in specific cell types and is synthesized constitutively in transformed B-cell lines. It also displays differential induction in response to tumor necrosis factor alpha, interleukin-1, or phorbol ester stimulation compared to I kappaB alpha in non-B-cell lines. Therefore, I kappaB epsilon represents a novel I kappaB family member which provides an alternative mechanism for regulation of NF-kappaB-dependent transcription.
PMCID: PMC232469  PMID: 9315679
12.  Transcriptional regulation of the human iNOS gene by IL-1beta in endothelial cells. 
Molecular Medicine  2001;7(5):329-343.
BACKGROUND: Vascular endothelium participates in the control of vascular tone and function via the release of nitric oxide (NO) by the endothelial-type NO synthase (eNOS). Inducible NO synthase (iNOS) expression in endothelial cells occurs in many clinical conditions following induction by lipopolysaccharide or cytokines and generates large quantities of NO that result in endothelial cell activation and dysfunction. No information exists on the transcriptional regulation of the human iNOS gene (or that of other species) in endothelial cells. MATERIALS AND METHODS: We examined the transcriptional regulation of the human iNOS gene by interleukin-1beta (IL-1beta) in rat pulmonary microvascular endothelial cells (PVEC) by transient cotransfections of different iNOS-promoter constructs and cDNA of different transcription factors and regulatory proteins. RESULTS: The -1034/+88 bp iNOS promoter was strongly induced by IL-1beta, the regulatory elements for such induction being localized downstream of -205 bp. Cotransfection experiments with NF-kappaB isoforms, IkappaB isoforms, and IKK mutants suggested that the NF-kappaB site at -115/-106 bp is important, but not sufficient, for induction of iNOS promoter and that the role of NF-kappaB is partially independent of its binding site. C/EBP sites within the -205/+88 bp region were shown to be responsible, along with NF-kappaB site, for induction of iNOS promoter by IL-1beta. Overexpression of C/EBPalpha, C/EBPdelta, and liver-enriched activator protein (LAP) activated the promoter, whereas overexpression of liver-enriched inhibitory protein (LIP) strongly suppressed it. C/EBPbeta (LAP and LIP isoforms) was constitutively present in PVEC and was induced (approximately 2-fold) by IL-1beta, whereas C/EBPdelta was not constitutively expressed but was strongly induced by IL-1beta. Both C/EBPbeta and C/EBPdelta participated in DNA-protein complex formation. CONCLUSION: Both NF-kappaB and C/EBP pathways are important for the transcriptional regulation of the human iNOS gene by IL-1beta in PVEC.
PMCID: PMC1950040  PMID: 11474579
13.  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
14.  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
15.  Synergistic activation of NF-kappaB by tumor necrosis factor alpha and gamma interferon via enhanced I kappaB alpha degradation and de novo I kappaBbeta degradation. 
Molecular and Cellular Biology  1997;17(11):6746-6754.
Tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) are required for an effective immune response to bacterial infection and these cytokines synergize in a variety of biological responses, including the induction of cytokine, cell adhesion, and inducible nitrous oxide synthase gene expression. Typically, the synergistic effect on gene expression is due to the independent activation of nuclear factor kappaB (NF-kappaB) by TNF-alpha and of signal transducers and activators of transcription or IFN-regulatory factor 1 by IFNs, allowing these transcription factors to bind their unique promoter sites. However, since activation of NF-kappaB by TNF-alpha is often transient and would not activate long-term kappaB-dependent transcription effectively, we explored the effects of IFN-gamma on TNF-alpha-induced NF-kappaB activity. IFN-gamma, which typically does not activate NF-kappaB, synergistically enhanced TNF-alpha-induced NF-kappaB nuclear translocation via a mechanism that involves the induced degradation of I kappaBbeta and that apparently requires tyrosine kinase activity in preneuronal cells but not in endothelial cells. Correspondingly, cotreatment of cells with TNF-alpha and IFN-gamma leads to persistent activation of NF-kappaB and to potent activation of kappaB-dependent gene expression, which may explain, at least in part, the synergy observed between these cytokines, as well as their involvement in the generation of an effective immune response.
PMCID: PMC232529  PMID: 9343439
16.  Modulation of the Osteosarcoma Expression Phenotype by MicroRNAs 
PLoS ONE  2012;7(10):e48086.
Osteosarcomas are the most common primary malignant tumors of bone and show multiple and complex genomic aberrations. miRNAs are non-coding RNAs capable of regulating gene expression at the post transcriptional level, and miRNAs and their target genes may represent novel therapeutic targets or biomarkers for osteosarcoma. In order to investigate the involvement of miRNAs in osteosarcoma development, global microarray analyses of a panel of 19 human osteosarcoma cell lines was performed.
Principal findings
We identified 177 miRNAs that were differentially expressed in osteosarcoma cell lines relative to normal bone. Among these, miR-126/miR-126*, miR-142-3p, miR-150, miR-223, miR-486-5p and members of the miR-1/miR-133a, miR-144/miR-451, miR-195/miR-497 and miR-206/miR-133b clusters were found to be downregulated in osteosarcoma cell lines. All miRNAs in the paralogous clusters miR-17-92, miR-106b-25 and miR-106a-92 were overexpressed. Furthermore, the upregulated miRNAs included miR-9/miR-9*, miR-21*, miR-31/miR-31*, miR-196a/miR-196b, miR-374a and members of the miR-29 and miR-130/301 families. The most interesting inversely correlated miRNA/mRNA pairs in osteosarcoma cell lines included miR-9/TGFBR2 and miR-29/p85α regulatory subunit of PI3K. PTEN mRNA correlated inversely with miR-92a and members of the miR-17 and miR-130/301 families. Expression profiles of selected miRNAs were confirmed in clinical samples. A set of miRNAs, miR-1, miR-18a, miR-18b, miR-19b, miR-31, miR-126, miR-142-3p, miR-133b, miR-144, miR-195, miR-223, miR-451 and miR-497 was identified with an intermediate expression level in osteosarcoma clinical samples compared to osteoblasts and bone, which may reflect the differentiation level of osteosarcoma relative to the undifferentiated osteoblast and fully differentiated normal bone. Significance: This study provides an integrated analysis of miRNA and mRNA in osteosarcoma, and gives new insight into the complex genetic mechanisms of osteosarcoma development and progression.
PMCID: PMC3485010  PMID: 23133552
17.  Chronic human immunodeficiency virus type 1 infection of myeloid cells disrupts the autoregulatory control of the NF-kappaB/Rel pathway via enhanced IkappaBalpha degradation. 
Journal of Virology  1996;70(8):5183-5193.
Productive human immunodeficiency virus type 1 (HIV-1) infection causes sustained NF-kappaB DNA-binding activity in chronically infected monocytic cells. A direct temporal correlation exists between HIV infection and the appearance of NF-kappaB DNA-binding activity in myelomonoblastic PLB-985 cells. To examine the molecular basis of constitutive NF-kappaB DNA-binding activity in HIV1 -infected cells, we analyzed the phosphorylation and turnover of IkappaBalpha protein, the activity of the double-stranded RNA-dependent protein kinase (PKR) and the intracellular levels of NF-kappaB subunits in the PLB-985 and U937 myeloid cell models. HIV-1 infection resulted in constitutive, low-level expression of type 1 interferon (IFN) at the mRNA level. Constitutive PKR activity was also detected in HIV-1-infected cells as a result of low-level IFN production, since the addition of anti-IFN-alpha/beta antibody to the cells decreased PKR expression. Furthermore, the analysis of IkappaBalpha turnover demonstrated an increased degradation of IkappaBalpha in HIV-1-infected cells that may account for the constitutive DNA binding activity. A dramatic increase in the intracellular levels of NF-kappaB subunits c-Rel and NF-kappaB2 p100 and a moderate increase in NF-kappaB2 p52 and RelA(p65) were detected in HIV-1-infected cells, whereas NF-kappaB1 p105/p50 levels were not altered relative to the levels in uninfected cells. We suggest that HIV-1 infection of myeloid cells induces IFN production and PKR activity, which in turn contribute to enhanced IkappaBalpha phosphorylation and subsequent degradation. Nuclear translocation of NF-kappaB subunits may ultimately increase the intracellular pool of NF-kappaB/IkappaBalpha by an autoregulatory mechanism. Enhanced turnover of IkappaBalpha and the accumulation of NF-kappaB/Rel proteins may contribute to the chronically activated state of HIV-1-infected cells.
PMCID: PMC190474  PMID: 8764027
18.  Dexamethasone prevents interleukin-1beta-induced nuclear factor-kappaB activation by upregulating IkappaB-alpha synthesis, in lymphoblastic cells. 
Mediators of Inflammation  2003;12(1):37-46.
AIMS: Glucocorticoids (GCs) exert some of their anti-inflammatory actions by preventing the activation of the transcription factor nuclear factor (NF)-kappaB. The GC-dependent inhibition of NF-kappaB may occur at different levels, but the mechanisms involved are still incompletely understood. In this work, we investigated whether the synthetic GC, dexamethasone (Dex), modulates the activity of NF-kappaB in the lymphoblastic CCRF-CEM cell line. We also evaluated the ability of Dex to prevent the activation of NF-kappaB in response to the potent proinflammatory cytokine, interleukin (IL)-1beta. RESULTS: Exposure of the cells to Dex (1 microM) induced the rapid degradation of IkappaB-alpha, leading to the transient translocation of the NF-kappaB family members p65 and p50 from the cytoplasm to the nucleus, as evaluated by western blot. Electrophoretic mobility shift assays revealed that, in the nucleus, these NF-kappaB proteins formed protein-DNA complexes, indicating a transient activation of NF-kappaB. Additionally, Dex also induced de novo synthesis of IkappaB-alpha, following its degradation. Finally, when the cells were exposed to Dex (1 microM) prior to stimulation with IL-1beta (20 ng/ml), Dex was efficient in preventing IL-1beta-induced NF-kappaB activation. The GC antagonist, RU 486 (10 microM), did not prevent any of the effects of Dex reported here. CONCLUSION: Our results indicate that, in CCRF-CEM cells, Dex prevents NF-kappaB activation, induced by IL-1beta, by a mechanism that involves the upregulation of IkappaB-alpha synthesis, and that depends on the early and transient activation of NF-kappaB.
PMCID: PMC1781587  PMID: 12745547
19.  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
20.  Distinct domains of IkappaB-alpha inhibit human immunodeficiency virus type 1 replication through NF-kappaB and Rev. 
Journal of Virology  1997;71(4):3161-3167.
Among the regulators of human immunodeficiency virus (HIV) replication is the cellular transcription factor NF-kappaB, whose activity is regulated through inhibition by IkappaB family members. We have shown previously that I kappaB-alpha inhibits HIV type 1 (HIV-1) replication, and unexpectedly, IkappaB-alpha was found both to suppress HIV-1 transcription and to inhibit Rev function. The relative contributions and specificities of these mechanisms to HIV replication were unknown. Here, we report that the region of IkappaB-alpha which blocks Rev function is separable from that required for inhibition of NF-kappaB. Molecular mutagenesis revealed that the N terminus of IkappaB-alpha is required for inhibition of Rev function, whereas mutants lacking the N terminus retained the ability to inhibit NF-kappaB function. Interestingly, the nuclear export sequence of IkappaB-alpha was not required for inhibition of Rev or NF-kappaB function in mammalian transfection assays. Conversely, the C terminus of IkappaB-alpha was not required for the inhibition of Rev, while deletion of this region resulted in a loss of NF-kappaB inhibition. Another IkappaB family member with a distinct amino-terminal sequence, IkappaB-beta, inhibited NF-kappaB but not Rev function. These studies indicate that the inhibition of Rev by IkappaB-alpha is independent of NF-kappaB. Mutants defective in inhibition of either Rev or NF-kappaB retained the ability to inhibit HIV-1 replication, suggesting that both functions may contribute to the inhibition of HIV replication by I kappaB-alpha.
PMCID: PMC191448  PMID: 9060679
21.  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.
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.
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.
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.
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.
PMCID: PMC3930545  PMID: 24529193
Esophageal squamous cell carcinoma; Gene regulation; NF-κB; Mcl-1; Cell viability
22.  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
23.  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
24.  Attenuation of nuclear factor kappa B (NF-kappaB) promotes apoptosis of kidney epithelial cells: a potential mechanism of mercury-induced nephrotoxicity. 
Environmental Health Perspectives  2002;110(Suppl 5):819-822.
Nuclear factor kappa B (NF-kappaB), a pleiotropic transcriptional factor that promotes cell survival and protects cells from apoptosis, requires reduced thiols at critical steps in its activation pathway. Mercuric ion (Hg(2+)), one of the strongest thiol-binding agents known, impairs NF-kappaB activation and transcriptional activity in normal rat kidney epithelial (NRK52E) cells at concentrations as low as 0.5 microM by binding to specific reduced thiol moieties in the NF-kappaB activation pathway. We hypothesized that prevention of NF-kappaB activation by Hg(2+) will increase the sensitivity of kidney cells to the apoptosis-inducing effects of other toxicants to which these cells are otherwise resistant by virtue of their NF-kappaB-activating capacity. Fewer than 5% of untreated kidney cells in culture (70-90% confluent) were found to be apoptotic when evaluated by DNA fragmentation (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling) or flow cytometric DNA profile analyses. Hg(2+) (5 microM) treatment for 24 hr increased this proportion by 1.5- to 2-fold. Neither lipopolysaccharide (LPS) (1 microg/mL) nor tumor necrosis factor-alpha (TNF-alpha; 300 U/mL), both potent activators of NF-kappaB in kidney cells, significantly altered the proportion of apoptotic cells, compared with untreated controls, when applied without Hg(2+) pretreatment. However, when LPS or TNF-alpha was administered after Hg(2+) pretreatment (5 microM for 30 min), the proportion of cells undergoing apoptosis 22 hr later increased by 4- to 6-fold compared with untreated controls. In contrast, Hg(2+) pretreatment did not increase the amount of apoptosis caused by apoptosis-inducing agents that do not activate NF-kappaB (staurosporine, Fas ligand). These findings suggest that Hg(2+) enhances the sensitivity of kidney cells to apoptotic stimuli as a consequence of inhibition of NF-kappaB activity. Because apoptosis is known to play a key role in the pathogenesis of renal failure resulting from toxicant injury to proximal tubular cells, promotion of apoptosis via inhibition of NF-kappaB activity may define a novel molecular mechanism by which Hg(2+) toxicity is initiated in kidney cells.
PMCID: PMC1241252  PMID: 12426138
25.  Coordinate transcription and V(D)J recombination of the kappa immunoglobulin light-chain locus: NF-kappaB-dependent and -independent pathways of activation. 
Molecular and Cellular Biology  1997;17(7):3477-3487.
To further elucidate the potential role of mitogens and cytokines in regulation of the kappa immunoglobulin light-chain locus, we have characterized the activation of transcription factor binding, kappa germ line transcription, DNase I hypersensitivity, and Vkappa-to-Jkappa recombination upon induction of model pre-B-cell lines. We find that both lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) are capable of activating germ line transcription, DNase I hypersensitivity, and recombination of the kappa locus. We also find that transforming growth factor beta is capable of completely inhibiting LPS activation of transcription and recombination but has no apparent effect on activation of transcription factor binding, including activation of NF-kappaB. To address the functional role of NF-kappaB in LPS and IFN-gamma induction of these events, we blocked the nuclear translocation of NF-kappaB by overexpression of a dominant negative mutant of IkappaB-alpha (IkappaB deltaN). Overexpression of the IkappaB deltaN protein results in an inhibition of LPS but not IFN-gamma activation of germ line transcription, DNase I hypersensitivity, and Vkappa-to-Jkappa recombination. Our results demonstrate that activation of NF-kappaB is necessary but not sufficient for LPS activation of transcription and recombination at kappa. These results also suggest that NF-kappaB is not required for IFN-gamma activation of transcription or recombination. These results are important in establishing that there are multiple independent pathways of activation of both transcription and recombination.
PMCID: PMC232201  PMID: 9199283

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