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Glioblastoma multiforme (GBM) carries a poor prognosis and continues to lack effective treatments. Glioblastoma stem cells (GSCs) drive tumor formation, invasion, and drug resistance and, as such, are the focus of studies to identify new therapies for disease control. Here, we identify the involvement of IKK and NF-κB signaling in the maintenance of GSCs. Inhibition of this pathway impairs self-renewal as analyzed in tumorsphere formation and GBM expansion as analyzed in brain slice culture. Interestingly, both the canonical and non-canonical branches of the NF-κB pathway are shown to contribute to this phenotype. One source of NF-κB activation in GBM involves the TGF-β/TAK1 signaling axis. Together, our results demonstrate a role for the NF-κB pathway in GSCs and provide a mechanistic basis for its potential as a therapeutic target in glioblastoma.

Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. These results argue that targeting downstream effectors of RAS will be an alternative route for blocking RAS-driven oncogenic pathways. We and others have shown that oncogenic RAS activates the NF-κB transcription factor pathway and that KRAS-induced lung tumorigenesis is suppressed by expression of a degradation-resistant form of the IκBα inhibitor or by genetic deletion of IKKβ or the RELA/p65 subunit of NF-κB. Here, genetic and pharmacological approaches were utilized to inactivate IKK in human primary lung epithelial cells transformed by KRAS, as well as KRAS mutant lung cancer cell lines. Administration of the highly specific IKKβ inhibitor Compound A (CmpdA) led to NF-κB inhibition in different KRAS mutant lung cells and siRNA-mediated knockdown of IKKα or IKKβ reduced activity of the NF-κB canonical pathway. Next, we determined that both IKKα and IKKβ contribute to oncogenic properties of KRAS mutant lung cells, particularly when p53 activity is disrupted. Based on these results, CmpdA was tested for potential therapeutic intervention in the Kras-induced lung cancer mouse model (LSL-Kras (G12D)) combined with loss of p53 (LSL-Kras (G12D)/p53 (fl/fl)). CmpdA treatment was well tolerated and mice treated with this IKKβ inhibitor presented smaller and lower grade tumors than mice treated with placebo. Additionally, IKKβ inhibition reduced inflammation and angiogenesis. These results support the concept of targeting IKK as a therapeutic approach for oncogenic RAS-driven tumors with altered p53 activity.

Glutamate is a critical neurotransmitter of the central nervous system (CNS) and also an important regulator of cell survival and proliferation. The binding of glutamate to metabotropic glutamate receptors induces signal transduction cascades that lead to gene-specific transcription. The transcription factor NF-kappaB, which regulates cell proliferation and survival, is activated by glutamate; however, the glutamate receptor-induced signaling pathways that lead to this activation are not clearly defined. Here we investigate the glutamate-induced activation of NF-kappaB in glial cells of the CNS, including primary astrocytes. We show that glutamate induces phosphorylation, nuclear accumulation, DNA binding, and transcriptional activation function of glial p65. The glutamate-induced activation of NF-kappaB requires calcium-dependent IkappaB kinase alpha (IKKalpha) and IKKbeta activation and induces p65-IkappaBalpha dissociation in the absence of IkappaBalpha phosphorylation or degradation. Moreover, glutamate-induced IKK preferentially targets the phosphorylation of p65 but not IkappaBalpha. Finally, we show that the ability of glutamate to activate NF-kappaB requires cross-coupled signaling with the epidermal growth factor receptor. Our results provide insight into a glutamate-induced regulatory pathway distinct from that described for cytokine-induced NF-kappaB activation and have important implications with regard to both normal glial cell physiology and pathogenesis.

While NF-kappaB is considered to play key roles in the development and progression of many cancers, the mechanisms whereby this transcription factor is activated in cancer are poorly understood. A key oncoprotein in a variety of cancers is the serine-threonine kinase Akt, which can be activated by mutations in PI3K, by loss of expression/activity of PTEN, or through signaling induced by growth factors and their receptors. A key effector of Akt-induced signaling is the regulatory protein mTOR (mammalian target of rapamycin). We show here that mTOR downstream from Akt controls NF-kappaB activity in PTEN-null/inactive prostate cancer cells via interaction with and stimulation of IKK. The mTOR-associated protein Raptor is required for the ability of Akt to induce NF-kappaB activity. Correspondingly, the mTOR inhibitor rapamycin is shown to suppress IKK activity in PTEN-deficient prostate cancer cells through a mechanism that may involve dissociation of Raptor from mTOR. The results provide insight into the effects of Akt/mTOR-dependent signaling on gene expression and into the therapeutic action of rapamycin.

Autoimmunity results from a breakdown in tolerance mechanisms that regulate autoreactive lymphocytes. We recently showed that during innate immune responses, secretion of IL-6 by dendritic cells (DCs) maintained autoreactive B cells in an unresponsive state. In this study, we describe that TLR4-activated DCs from lupus-prone mice are defective in repressing autoantibody secretion, coincident with diminished IL-6 secretion. Reduced secretion of IL-6 by MRL/lpr DCs reflected diminished synthesis and failure to sustain IL-6 mRNA production. This occurred coincident with lack of NF-kappaB and AP-1 DNA binding and failure to sustain IkappaBalpha phosphorylation. Analysis of individual mice showed that some animals partially repressed Ig secretion despite reduced levels of IL-6. This suggests that in addition to IL-6, DCs secrete other soluble factor(s) that regulate autoreactive B cells. Collectively, the data show that MRL/lpr mice are defective in DC/IL-6-mediated tolerance, but that some individuals maintain the ability to repress autoantibody secretion by an alternative mechanism.

Loss of glycogen synthase kinase 3beta (GSK-3beta) in mice results in embryonic lethality via hepatocyte apoptosis. Consistent with this result, cells from these mice have diminished nuclear factor kappaB (NF-kappaB) activity, implying a functional role for GSK-3beta in regulating NF-kappaB. Here, we have explored mechanisms by which GSK-3beta may control NF-kappaB function. We show that cytokine-induced IkappaB kinase activity and subsequent phosphorylation of IkappaBalpha, p105, and p65 are not affected by the absence of GSK-3beta activity. Furthermore, nuclear accumulation of p65 following tumor necrosis factor treatment is unaffected by the loss of GSK-3beta. However, NF-kappaB DNA binding activity is reduced in GSK-3beta null cells and in cells treated with a pharmacological inhibitor of GSK-3. Expression of certain NF-kappaB-regulated genes, such as IkappaBalpha and macrophage inflammatory protein 2, is minimally affected by the absence of GSK-3beta. Conversely, we have identified a subset of NF-kappaB-regulated genes, including those for interleukin-6 and monocyte chemoattractant protein 1, that require GSK-3beta for efficient expression. We show that efficient localization of p65 to the promoter regions of the interleukin-6 and monocyte chemoattractant protein 1 genes following tumor necrosis factor alpha treatment requires GSK-3beta. Therefore, GSK-3beta has profound effects on transcription in a gene-specific manner through a mechanism involving control of promoter-specific recruitment of NF-kappaB.

Mitogenic activation of expression of immediate-early genes, such as c-fos, is controlled through signal-induced phosphorylation of constitutively bound transcription factors that is correlated with a nucleosomal response that involves inducible chromatin modifications, such as histone phosphorylation and acetylation. Here we have explored a potential role for the transcription factor NF-kappaB and its associated signaling components in mediating induction of c-fos gene expression downstream of epidermal growth factor (EGF)-dependent signaling. Here we show that EGF treatment of quiescent fibroblast does not induce the classical pathway of NF-kappaB activation through IkappaB kinase (IKK)-directed IkappaBalpha phosphorylation. Interestingly, efficient induction of c-fos transcription requires IKKalpha, one of the subunits of the IkappaB kinase complex. The NF-kappaB subunit, p65/RelA, is found constitutively associated with the c-fos promoter, and knock-out of this transcription factor significantly reduces c-fos gene expression. Importantly, EGF induces the recruitment of IKKalpha to the c-fos promoter to regulate promoter-specific histone H3 Ser(10) phosphorylation in a manner that is independent of p65/RelA. Collectively, our data demonstrate that IKKalpha and p65/RelA contribute significantly to EGF-induced c-fos gene expression in a manner independent of the classical, IkappaBalpha degradation, p65/RelA nuclear accumulation response pathway.

Cytokines, such as tumor necrosis factor-alpha (TNFalpha), potently inhibit the differentiation of mesenchymal cells and down-regulate the expression of Sox9 and MyoD, transcription factors required for chondrocyte and myocyte development. Previously, we demonstrated that NF-kappaB controls TNFalpha-mediated suppression of myogenesis through a mechanism involving MyoD mRNA down-regulation. Here, we show that NF-kappaB also suppresses chondrogenesis and destabilizes Sox9 mRNA levels. Multiple copies of an mRNA cis-regulatory motif (5'-ACUACAG-3') are necessary and sufficient for NF-kappaB-mediated Sox9 and MyoD down-regulation. Thus, in response to cytokine signaling, NF-kappaB modulates the differentiation of mesenchymal-derived cell lineages via RNA sequence-dependent, posttranscriptional down-regulation of key developmental regulators.

NF-kappaB is a principal transcriptional regulator of diverse cytokine-mediated processes and is tightly controlled by the IkappaB kinase complex (IKK-alpha/beta/gamma). IKK-beta and IKK-gamma are critical for cytokine-induced NF-kappaB function, whereas IKK-alpha is thought to be involved in other regulatory pathways. However, recent data suggest a role for IKK-alpha in NF-kappaB-dependent gene expression in response to cytokine treatment. Here we demonstrate nuclear accumulation of IKK-alpha after cytokine exposure, suggesting a nuclear function for this protein. Consistent with this, chromatin immunoprecipitation (ChIP) assays reveal that IKK-alpha was recruited to the promoter regions of NF-kappaB-regulated genes on stimulation with tumour-necrosis factor-alpha. Notably, NF-kappaB-regulated gene expression is suppressed by the loss of IKK-alpha and this correlates with a complete loss of gene-specific phosphorylation of histone H3 on serine 10, a modification previously associated with positive gene expression. Furthermore, we show that IKK-alpha can directly phosphorylate histone H3 in vitro, suggesting a new substrate for this kinase. We propose that IKK-alpha is an essential regulator of NF-kappaB-dependent gene expression through control of promoter-associated histone phosphorylation after cytokine exposure. These findings provide additional insight into the role of the IKK complex in NF-kappaB-regulated gene expression.

The transcription factor NF-kappa B has been shown to be predominantly cytoplasmically localized in the absence of an inductive signal. Stimulation of cells with inflammatory cytokines such as tumor necrosis factor alpha or interleukin-1 induces the degradation of I kappa B, the inhibitor of NF-kappa B, allowing nuclear accumulation of NF-kappa B and regulation of specific gene expression. The degradation of I kappa B is controlled initially by phosphorylation induced by the I kappa B kinase, which leads to ubiquitination and subsequent proteolysis of the inhibitor by the proteasome. We report here that NF-kappa B and I kappa B alpha (but not I kappa B beta) are also localized in the mitochondria. Stimulation of cells with tumor necrosis factor alpha leads to the phosphorylation of mitochondrial I kappa B alpha and its subsequent degradation by a nonproteasome-dependent pathway. Interestingly, expression of the mitochondrially encoded cytochrome c oxidase III and cytochrome b mRNAs were reduced by cytokine treatment of cells. Inhibition of activation of mitochondrial NF-kappa B by expression of the superrepressor form of I kappa B alpha inhibited the loss of expression of both cytochrome c oxidase III and cytochrome b mRNA. These data indicate that the NF-kappa B regulatory pathway exists in mitochondria and that NF-kappa B can negatively regulate mitochondrial mRNA expression.

Members of the NF-kappa B/Rel transcription factor family have been shown recently to be required for cellular transformation by oncogenic Ras and by other oncoproteins and to suppress transformation-associated apoptosis. Furthermore, NF-kappa B has been shown to be activated by several oncoproteins including HER2/Neu, a receptor tyrosine kinase often expressed in human breast cancer. Human breast cancer cell lines, human breast tumors and normal adjacent tissue were analysed by gel mobility shift assay, immunoblotting of nuclear extracts and immunohistochemistry for activation of NF-kappa B. Furthermore, RNA levels for NF-kappa B-activated genes were analysed in order to determine if NF-kappa B is functionally active in human breast cancer. Our data indicate that the p65/RelA subunit of NF-kappa B is activated (i.e., nuclear) in breast cancer cell lines. However, breast tumors exhibit an absence or low level of nuclear p65/RelA but show activated c-Rel, p50 and p52 as compared to nontumorigenic adjacent tissue. Additionally, the I kappa B homolog Bcl-3, which functions to stimulate transcription with p50 or p52, was also activated in breast tumors. There was no apparent correlation between estrogen receptor status and levels of nuclear NF-kappa B complexes. Transcripts of NF-kappa B-regulated genes were found elevated in breast tumors, as compared to adjacent normal tissue, indicating functional NF-kappa B activity. These data suggest a potential role for a subset of NF-kappa B and I kappa B family proteins, particularly NF-kappa B/p52 and Bcl-3, in human breast cancer. Additionally, the activation of functional NF-kappa B in these tumors likely involves a signal transduction pathway distinct from that utilized by cytokines.

The ras proto-oncogene is frequently mutated in human tumors and functions to chronically stimulate signal transduction cascades resulting in the synthesis or activation of specific transcription factors, including Ets, c-Myc, c-Jun, and nuclear factor kappa B (NF-kappaB). These Ras-responsive transcription factors are required for transformation, but the mechanisms by which these proteins facilitate oncogenesis have not been fully established. Oncogenic Ras was shown to initiate a p53-independent apoptotic response that was suppressed through the activation of NF-kappaB. These results provide an explanation for the requirement of NF-kappaB for Ras-mediated oncogenesis and provide evidence that Ras-transformed cells are susceptible to apoptosis even if they do not express the p53 tumor-suppressor gene product.

NF-kappa B is an important transcription factor required for T cell proliferation and other immunological functions. The NF-kappa B1 gene encodes a 105-kD protein that is the precursor of the p50 component of NF-kappa B. Previously, we and others have demonstrated that NF-kappa B regulates the NF-kappa B1 gene. In this manuscript we have investigated the molecular mechanisms by which T cell lines stimulated with phorbol 12-myristate 13-acetate (PMA) and phytohemagglutin (PHA) display significantly higher levels of NF-kappa B1 encoding transcripts than cells stimulated with tumor necrosis factor-alpha, despite the fact that both stimuli activate NF-kappa B. Characterization of the NF-kappa B1 promoter identified an Egr-1 site which was found to be essential for both the PMA/PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-kappa B and Egr-1. Furthermore, Egr-1 induction was required for endogenous NF-kappa B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-kappa B1 transcription. Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-kappa B may have important ramifications in T cell development by upregulating NF-kappa B1 gene expression.

Tat protein of HIV-1 is a potent transactivator of transcription and essential for HIV-1 replication. In addition, Tat has been proposed to possess immunosuppressive functions, suggesting that Tat may play a direct role in the immune dysfunction associated with AIDS. Recently, it has been reported that Tat represses activity of a major histocompatibility complex (MHC) class I gene promoter. Because HIV infection downmodulates expression of class I molecules, this data strongly suggests that Tat downregulates class I expression and leads to loss of CTL activity. Here, we report effects of Tat on class I expression using a human cell line, T0, expressing Tat (TO-Tat). Northern blot analysis shows that levels of MHC class I transcripts are normal in T0-Tat. Flow cytometry analyses indicate that expression of HLA class I molecules is not substantially downregulated to any great extent by Tat in T0-Tat. Further, pulse-chase experiments followed by Endoglycosidase-H treatment show that the rate of maturation and processing of class I molecules in T0-Tat is indistinguishable from that in the original cell line, T0. Taken together, these data suggest that Tat expression does not necessarily result in downregulation of class I expression.

Glucocorticoids are potent immunosuppressive drugs, but their mechanism is poorly understood. Nuclear factor kappa B (NF-kappa B), a regulator of immune system and inflammation genes, may be a target for glucocorticoid-mediated immunosuppression. The activation of NF-kappa B involves the targeted degradation of its cytoplasmic inhibitor, I kappa B alpha, and the translocation of NF-kappa B to the nucleus. Here it is shown that the synthetic glucocorticoid dexamethasone induces the transcription of the I kappa B alpha gene, which results in an increased rate of I kappa B alpha protein synthesis. Stimulation by tumor necrosis factor causes the release of NF-kappa B from I kappa B alpha. However, in the presence of dexamethasone this newly released NF-kappa B quickly reassociates with newly synthesized I kappa B alpha, thus markedly reducing the amount of NF-kappa B that translocates to the nucleus. This decrease in nuclear NF-kappa B is predicted to markedly decrease cytokine secretion and thus effectively block the activation of the immune system.

NF-kappa B and C/EBP represent distinct families of transcription factors that target unique DNA enhancer elements. The heterodimeric NF-kappa B complex is composed of two subunits, a 50- and a 65-kDa protein. All members of the NF-kappa B family, including the product of the proto-oncogene c-rel, are characterized by their highly homologous approximately 300-amino-acid N-terminal region. This Rel homology domain mediates DNA binding, dimerization, and nuclear targeting of these proteins. C/EBP contains the bZIP region, which is characterized by two motifs in the C-terminal half of the protein: a basic region involved in DNA binding and a leucine zipper motif involved in dimerization. The C/EBP family consist of several related proteins, C/EBP alpha, C/EBP beta, C/EBP gamma, and C/EBP delta, that form homodimers and that form heterodimers with each other. We now demonstrated the unexpected cross-coupling of members of the NF-kappa B family three members of the C/EBP family. NF-kappa B p65, p50, and Rel functionally synergize with C/EBP alpha, C/EBP beta, and C/EBP delta. This cross-coupling results in the inhibition of promoters with kappa B enhancer motifs and in the synergistic stimulation of promoters with C/EBP binding sites. These studies demonstrate that NF-kappa B augments gene expression mediated by a multimerized c-fos serum response element in the presence of C/EBP. We show a direct physical association of the bZIP region of C/EBP with the Rel homology domain of NF-kappa B. The cross-coupling of NF-kappa B with C/EBP highlights a mechanism of gene regulation involving an interaction between distinct transcription factor families.

Activation of the murine c-myc promoter by murine c-Myb protein was examined in several cell lines by using a transient expression system in which Myb expression vectors activate the c-myc promoter linked to a chloramphenicol acetyltransferase reporter gene or a genomic beta-globin gene. S1 nuclease protection analyses confirmed that the induction of c-myc by c-Myb was transcriptional and affected both P1 and P2 start sites in a murine T-cell line, EL4, and a myelomonocytic line, WEHI-3. Mutational analyses of the c-myc promoter revealed that two distinct regions could confer Myb responsiveness in two T-cell lines, a distal site upstream of P1 and a proximal site within the first noncoding exon. In contrast, only the proximal site was required for other cell lineages examined. Five separate Myb-binding sites were located in this proximal site and found to be important for c-Myb trans activation. DNA binding was necessary for c-myc activation, as shown by the loss of function associated with mutation of Myb's DNA-binding domain and by trans-dominant repressor activity of the DNA binding, trans-activation-defective mutant. The involvement of additional protein factors was addressed by inhibiting protein synthesis with cycloheximide in a conditional expression system in which the activity of presynthesized Myb was under the control of estrogen. These experiments indicate that de novo synthesis of additional proteins was not necessary for c-myc trans activation. Together these data reveal two cell lineage-dependent pathways by which c-Myb regulates c-myc; however, both pathways are mechanistically indistinguishable in that direct DNA binding by Myb is required for activating c-myc whereas neither de novo protein synthesis nor other labile proteins are necessary.

NF-kappa B is a transcription factor involved in the regulation of numerous genes encoding proteins involved in immune function, in inflammation or in cellular growth control. NF-kappa B is typically characterized as a heterodimer of a 50-kDa subunit (p50) and a 65-kDa (p65) subunit. Interestingly, the p50 subunit is derived by processing of a 105-kDa precursor. Induction of NF-kappa B DNA-binding activity involves both the release of cytoplasmically stored factor from its inhibitor known as I kappa B and the induction of NF-kappa B gene expression. We report here the cloning and functional analysis of the promoter of the p50/p105 NF-kappa B gene. Our data suggest the existence of multiple transcription initiation sites for this gene in the B cell line Raji, Jurkat T cells, and HeLa cells. The promoter is constitutively active in these cells and is inducible by phorbol ester and mitogen stimulation of Jurkat T cells. Expression of I kappa B inhibits this inducible activation of the p50/p105 promoter. Furthermore, we have shown that co-transfection of a p50/p105 promoter-reporter plasmid with expression vectors encoding the p50 or p65 subunits of NF-kappa B or c-Rel results in stimulation of gene expression. Supportive of the transfection data, we have identified a DNA-binding site for NF-kappa B in the promoter of the p50/p105 gene that is responsive only to a combination of p50 and p65. The data demonstrate that the p50/p105 NF-kappa B gene is regulated by members of the NF-kappa B/Rel family and likely by other important transcription factors.

Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type III and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.