Apoptosis promotes a caspase-induced amino-terminal truncation of IκBα that functions as a stable inhibitor of NF-kB

Caspases are cell death cysteine proteases that are activated upon the induction of the apoptotic program and cleave target proteins in a sequence- specific manner to promote cell death. Recently, Barkett et al. (Barkett, M., Xue, D., Horvitz, H. R., and Gilmore, T. D. (1997) J. BioL Chem. 272, 29419- 29422) have shown that IκBα, the inhibitory subunit of the transcription factor NF-κB, can be cleaved by caspase-3 in vitro at a site that potentially produces a dominant inhibitory form of IκBα. The involvement of NF-κB in the inhibition of cell death led us to ask whether apoptotic stimuli would induce the caspase-mediated cleavage of IκBα in vivo. In this study, we show that apoptosis leads to the caspase-mediated amino-terminal truncation of IκBα (δN-IκBα). Our data show that δN-IκBα can bind NF- κB, suppress NF-κB activation, and sensitize cells to death. Since activated NF-κB plays a role in the inhibition of cell death, these data suggest that caspase-mediated cleavage of IκBα may be a mechanism to suppress NF-κB and its associated antiapoptotic activity

Activation of nuclear factor-κB-dependent transcription by tumor necrosis factor-α is mediated through phosphorylation of RelA/p65 on serine 529

Nuclear factor-κB (NF-κB) is an essential transcription factor in the control of expression of genes involved in immune and inflammatory responses. In unstimulated cells, NF-κB complexes are sequestered in the cytoplasm through interactions with IκBα and other IκB proteins. Extracellular stimuli that activate NF-κB, such as tumor necrosis factor α (TNFα), cause rapid phosphorylation of IκBα at serines 32 and 36. The inducible phosphorylation of IκBα is followed by its ubiquitination and degradation, allowing NF-κB complexes to translocate into the nucleus and to activate gene expression. Previously, it has been shown that TNFα as well as other stimuli also lead to the phosphorylation of the RelA/p65 subunit of NF-κB. In this report, we demonstrate that the TNFα-induced phosphorylation of the RelA/p65 subunit occurs on serine 529, which is in the C-terminal (TA1) transactivation domain. Accordingly, the TNFα-induced phosphorylation of Rel/p65 increases NF-κB transcriptional activity but does not affect nuclear translocation or DNA binding affinity

NF-κB mediates inhibition of mesenchymal cell differentiation through a posttranscriptional gene silencing mechanism

Cytokines, such as tumor necrosis factor-α (TNFα), 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-κB controls TNFα-mediated suppression of myogenesis through a mechanism involving MyoD mRNA down-regulation. Here, we show that NF-κB 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-κB-mediated Sox9 and MyoD down-regulation. Thus, in response to cytokine signaling, NF-κB modulates the differentiation of mesenchymal-derived cell lineages via RNA sequence-dependent, posttranscriptional down-regulation of key developmental regulators

Involvement of double-stranded RNA-activated protein kinase in the synergistic activation of nuclear factor-κB by tumor necrosis factor-α and γ-interferon in preneuronal cells

Tumor necrosis factor-α (TNF-α) and γ-interferon (IFN-γ) cooperate during a variety of biological responses and ultimately synergistically enhance the expression of genes involved in immune and inflammatory responses. Recently, we demonstrated that IFN-γ can significantly potentiate TNF-α-induced nuclear factor (NF)-κB nuclear translocation in neuronal derived and endothelial cell lines. The mechanism by which these two cytokines exert their synergistic effect on NF-κB involves the de novo degradation of the NF-κB inhibitor, IκBβ. The double-stranded RNA- dependent kinase PKR is IFN-inducible and has been implicated in the activation of NF-κB; therefore, we examined the possibility that PKR may play a role in the synergistic activation of NF-κB during TNF-α/IFN-γ cotreatment. The PKR inhibitor 2-aminopurine (2-AP) inhibited TNF-α/IFN-γ- induced NF-κB nuclear translocation in neuronal derived cells but not in endothelial cells. The induced degradation of IκBβ, which is normally observed upon TNF-α/IFN-γ cotreatment, was blocked completely by 2-AP in neuronal derived cells. Also, 2-AP treatment or overexpression of a catalytically inactive PKR inhibited the TNF-α/IFN-γ-induced synergistic activation of κB-dependent gene expression. Our results suggest that the signal generated by IFN-γ, during TNF-α/IFN-γ cotreatment may require PKR to elicit enhanced NF-κB activity, and this signal may affect the stability of the IκBβ protein

Opposing Regulation of Choline Deficiency-induced Apoptosis by p53 and Nuclear Factor κB

We have previously shown that fetal rat brain cells, preneuronal (PC12), and hepatocyte (CWSV-1) cells undergo apoptosis during choline deficiency (CD). The PC12 and epithelial cell culture models were used to determine the molecular mechanism by which CD induces apoptosis. Our data indicate that CD leads to both growth arrest and apoptosis in a subpopulation of cells, which correlate with the up-regulation of the tumor suppressor protein p53 and concurrent up-regulation of the cyclin-dependent kinase-inhibitor p21 WAF1/CIP1. Additionally, CD induced both a G1/S and a G2/M arrest. Transient transfection of a dominant negative p53 (p53DN) construct into PC12 cells, which inhibited endogenous p53 activation, significantly reduced the induction of apoptosis associated with CD. Interestingly, CD also induced the persistent activation of the transcription factor NF-κB. Activation of NF-κB has been shown to promote cell survival and proposed to antagonize p53. Consistent with this, expression of a superrepressor form of IκBα (SR-IκBα) that functions to strongly inhibit NF-κB activation, profoundly enhanced cell death during CD. In summary, these results suggest that the effects of CD on apoptosis and subsequent cell survival are mediated through two different signaling pathways, p53 and NF-κB, respectively. Taken together, our data demonstrates the induction of opposing mechanisms associated with nutrient deficiency that may provide a molecular mechanism by which CD promotes carcinogenesis

Structure and evolution of the human IKBA gene

IκBα belongs to a gene family whose members are characterized by their 6-7 Ankyrin repeats, which allow them to interact with members of the Rel family of transcription factors. We have sequenced a human IκBα genomic clone to determine its gene structure. The human IκBα gene (IKBA) has six exons and five introns that span approximately 3.5 kb. This genomic organization is similar to that of other members of the Ankyrin gene family. The humanIKBAgene shares similar intron/exon boundaries with the humanBCL3andNFKB2genes, which is consistent with their conserved Ankyrin repeats. To examine further the evolutionary relationship between human IκBα and other members of its gene family, we performed a phylogenetic analysis. Although the resulting phylogenetic tree does not identify a common ancestor of the IκBα gene family, it indicates that this family diverges into two groups based on structure and function

Inhibition of NF-κB Activity by Thalidomide through Suppression of IκB Kinase Activity

The sedative and anti-nausea drug thalidomide, which causes birth defects in humans, has been shown to have both anti-inflammatory and anti-oncogenic properties. The anti-inflammatory effect of thalidomide is associated with suppression of cytokine expression and the anti-oncogenic effect with inhibition of angiogenesis. It is presently unclear whether the teratogenic properties of thalidomide are connected in any way to the beneficial, anti-disease characteristics of this drug. The transcription factor NF-κB has been shown to be a key regulator of inflammatory genes such as tumor necrosis factor-α and interleukin-8. Inhibition of NF-κB is associated with reduced inflammation in animal models, such as those for rheumatoid arthritis. We show here that thalidomide can block NF-κB activation through a mechanism that involves the inhibition of activity of the IκB kinase. Consistent with the observed inhibition of NF-κB, thalidomide blocked the cytokine-induced expression of NF-κB-regulated genes such as those encoding interleukin-8, TRAF1, and c-IAP2. These data indicate that the therapeutic potential for thalidomide may be based on its ability to block NF-κB activation through suppression of IκB kinase activity

Activation and repression of mammalian gene expression by the c-myc protein.

One mechanism by which nuclear-localized oncogenes might transform cells is through an ability to regulate gene expression. We show that the c-myc protein stimulates the level of appropriately initiated expression from the human heat shock protein 70 (hsp70) promoter. Sequences required for full activation lie upstream of the transcription initiation site and are distinct from sequences necessary for basal expression. These sequences also appear distinct from promoter sequences necessary for heat induction, serum induction, and induction by the papovavirus T antigens. The c-myc protein inhibits appropriately initiated expression from the mouse metallothionein I (MT-I) promoter. A mutation that removes 138 amino acids of exon 2 produces a c-myc gene product that is capable of activating the hsp70 promoter but is no longer capable of inhibiting MT-I expression, suggesting that these two properties reside in different domains of the c-myc protein. Expression from the adenovirus EII promoter is slightly inhibited, while expression from the SV40 early promoter is minimally affected by the c-myc protein. Both the spectrum of promoters regulated by the c-myc protein and the sequence requirements for that regulation differ from those of previously characterized viral trans-activating proteins. The data suggest that the c-myc protein can both stimulate and inhibit transcription from mammalian promoters in a novel manner

Interleukin-10 signaling blocks inhibitor of κB kinase activity and nuclear factor κB DNA binding

The transcription factor nuclear factor κB (NF-κB) coordinates the activation of numerous genes in response to pathogens and proinflammatory cytokines and is, therefore, pivotal in the development of acute and chronic inflammatory diseases. In its inactive state, NF-κB is constitutively present in the cytoplasm as a p50-p65 heterodimer bound to its inhibitory protein IκB. Proinflammatory cytokines, such as tumor necrosis factor (TNF), activate NF-κB by stimulating the activity of the IκB kinases (IKKs) which phosphorylate IκBα on serine residues 32 and 36, targeting it for rapid degradation by the 26 S proteasome. This enables the release and nuclear translocation of the NF-κB complex and activation of gene transcription. Interleukin-10 (IL-10) is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines which are known to be transcriptionally controlled by NF-κB. Conflicting data exists on the effects of IL-10 on TNF- and LPS-induced NF-≃B activity in human monocytes and the molecular mechanisms involved have not been elucidated. In this study, we show that IL-10 functions to block NF-≃B activity at two levels: 1) through the suppression of IKK activity and 2) through the inhibition of NF-κB DNA binding activity. This is the first evidence of an anti-inflammatory protein inhibiting IKK activity and demonstrates that IKK is a logical target for blocking inflammatory diseases

Tumor Necrosis Factor α-induced Phosphorylation of RelA/p65 on Ser 529 Is Controlled by Casein Kinase II

Nuclear factor kappaB (NF-kappaB)/Rel transcription factors are key regulators of a variety of genes involved in immune and inflammatory responses, growth, differentiation, apoptosis, and development. In unstimulated cells, NF-kappaB/Rel proteins are sequestered in the cytoplasm by IkappaB inhibitor proteins. Many extracellular stimuli, such as tumor necrosis factor alpha (TNFalpha), cause rapid phosphorylation of IkappaB at N-terminal serine residues leading to ubiquitination and degradation of the inhibitor. Subsequently, NF-kappaB proteins translocate to the nucleus and activate gene expression through kappaB response elements. TNFalpha, as well as certain other stimuli, also induces the phosphorylation of the NF-kappaB proteins. Previously, we have shown that TNFalpha induces RelA/p65 phosphorylation at serine 529 and that this inducible phosphorylation increases NF-kappaB transcriptional activity on an exogenously supplied reporter (). In this report, we demonstrate that casein kinase II (CKII) interacts with p65 in vivo and can phosphorylate p65 at serine 529 in vitro. A CKII inhibitor (PD144795) inhibited TNFalpha-induced p65 phosphorylation in vivo. Furthermore, our results indicate that the association between IkappaBalpha and p65 inhibits p65 phosphorylation by CKII and that degradation of IkappaBalpha allows CKII to phosphorylate p65 to increase NF-kappaB transactivation potential. These data may explain the ability of CKII to modulate cell growth and demonstrate a mechanism whereby CKII can function in an inducible manner