Patricia C. Cogswell

Role of Transcriptional Activation of IκBα in Mediation of Immunosuppression by Glucocorticoids

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

axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase.

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.

Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZIP interaction.

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.

Selective activation of NF-κB subunits in human breast cancer: potential roles for NF-κB2/p52 and for Bcl-3

Members of the NF-κB/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-κB 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-κB. Furthermore, RNA levels for NF-κB-activated genes were analysed in order to determine if NF-κB is functionally active in human breast cancer. Our data indicate that the p65/RelA subunit of NF-κB 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 IκB 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-κB complexes. Transcripts of NF-κB-regulated genes were found elevated in breast tumors, as compared to adjacent normal tissue, indicating functional NF-κB activity. These data suggest a potential role for a subset of NF-κB and IκB family proteins, particularly NF-κB/p52 and Bcl-3, in human breast cancer. Additionally, the activation of functional NF-κB in these tumors likely involves a signal transduction pathway distinct from that utilized by cytokines.

Akt-dependent regulation of NF-κB is controlled by mTOR and Raptor in association with IKK

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.

A nucleosomal function for IκB kinase-α in NF-κB-dependent gene expression

NF-κB is a principal transcriptional regulator of diverse cytokine-mediated processes and is tightly controlled by the IκB kinase complex (IKK-α/β/γ). IKK-β and IKK-γ are critical for cytokine-induced NF-κB function, whereas IKK-α is thought to be involved in other regulatory pathways. However, recent data suggest a role for IKK-α in NF-κB-dependent gene expression in response to cytokine treatment. Here we demonstrate nuclear accumulation of IKK-α after cytokine exposure, suggesting a nuclear function for this protein. Consistent with this, chromatin immunoprecipitation (ChIP) assays reveal that IKK-α was recruited to the promoter regions of NF-κB-regulated genes on stimulation with tumour-necrosis factor-α. Notably, NF-κB-regulated gene expression is suppressed by the loss of IKK-α 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-α can directly phosphorylate histone H3 in vitro, suggesting a new substrate for this kinase. We propose that IKK-α is an essential regulator of NF-κB-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-κB-regulated gene expression.

A nucleosomal function for IkappaB kinase-alpha in NF-kappaB-dependent gene expression.

NF-κB is a principal transcriptional regulator of diverse cytokine-mediated processes and is tightly controlled by the IκB kinase complex (IKK-α/β/γ). IKK-β and IKK-γ are critical for cytokine-induced NF-κB function, whereas IKK-α is thought to be involved in other regulatory pathways. However, recent data suggest a role for IKK-α in NF-κB-dependent gene expression in response to cytokine treatment. Here we demonstrate nuclear accumulation of IKK-α after cytokine exposure, suggesting a nuclear function for this protein. Consistent with this, chromatin immunoprecipitation (ChIP) assays reveal that IKK-α was recruited to the promoter regions of NF-κB-regulated genes on stimulation with tumour-necrosis factor-α. Notably, NF-κB-regulated gene expression is suppressed by the loss of IKK-α 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-α can directly phosphorylate histone H3 in vitro, suggesting a new substrate for this kinase. We propose that IKK-α is an essential regulator of NF-κB-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-κB-regulated gene expression.

Glycogen Synthase Kinase 3β Functions To Specify Gene-Specific, NF-κB-Dependent Transcription

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

CCAAT box binding protein NF-Y facilitates in vivo recruitment of upstream DNA binding transcription factors.

NF‐Y binds a CCAAT motif found in many eukaryotic polymerase II‐dependent promoters. In the HLA‐DRA promoter it has been demonstrated that stereo‐specific alignment between this motif and the upstream elements X1 and X2 is required for activation. To study the underlying mechanism for this requirement, a panel of transfected cell lines that maintained integrated, wild‐type and mutant promoters were analyzed by in vivo genomic footprinting. Cell lines harboring a mutated CCAAT element exhibited a loss of interactions at the CCAAT site, as expected, and no transcriptional activity. Most importantly, mutation of the CCAAT sequence nearly abolished in vivo binding at the X1 and X2 sites, while mutations of X1 and X2 had little effect on CCAAT box binding. However, X1 and X2 binding was interdependent. In vitro, X1 binding activities are known to be stabilized by NF‐Y binding. Interaction between NF‐Y and X box binding proteins was demonstrated by reciprocal co‐immunoprecipitation in the absence of DNA and co‐affinity purification in the presence of DNA. Collectively, these studies indicate that occupancy of the CCAAT element represents an early event affecting other protein‐DNA interactions and suggest that NF‐Y stabilizes and interacts with X box factors to mediate this function. These findings may represent a common theme among promoters containing a CCAAT element.

NF-κB and IκBα Are Found in the Mitochondria EVIDENCE FOR REGULATION OF MITOCHONDRIAL GENE EXPRESSION BY NF-κB

The transcription factor NF-κB 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 α or interleukin-1 induces the degradation of IκB, the inhibitor of NF-κB, allowing nuclear accumulation of NF-κB and regulation of specific gene expression. The degradation of IκB is controlled initially by phosphorylation induced by the IκB kinase, which leads to ubiquitination and subsequent proteolysis of the inhibitor by the proteasome. We report here that NF-κB and IκBα (but not IκBβ) are also localized in the mitochondria. Stimulation of cells with tumor necrosis factor α leads to the phosphorylation of mitochondrial IκBα and its subsequent degradation by a nonproteasome-dependent pathway. Interestingly, expression of the mitochondrially encoded cytochromec oxidase III and cytochrome bmRNAs were reduced by cytokine treatment of cells. Inhibition of activation of mitochondrial NF-κB by expression of the superrepressor form of IκBα inhibited the loss of expression of both cytochromec oxidase III and cytochrome b mRNA. These data indicate that the NF-κB regulatory pathway exists in mitochondria and that NF-κB can negatively regulate mitochondrial mRNA expression.