Amer A. Beg

Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation.

Nuclear factor kappa B (NF-kappa B) is a critical regulator of several genes which are involved in immune and inflammation responses. NF-kappa B, consisting of a 50-kDa protein (p50) and a 65-kDa protein (p65), is bound to a cytoplasmic retention protein called I kappa B. Stimulation of cells with a variety of inducers, including cytokines such as tumor necrosis factor and interleukin-1, leads to the activation and the translocation of p50/65 NF-kappa B into the nucleus. However, the in vivo mechanism of the activation process remains unknown. Here, we provide the first evidence that the in vivo mechanism of NF-kappa B activation is through the phosphorylation and subsequent loss of its inhibitor, I kappa B alpha. We also show that both I kappa B alpha loss and NF-kappa B activation are inhibited in the presence of antioxidants, demonstrating that the loss of I kappa B alpha is a prerequisite for NF-kappa B activation. Finally, we demonstrate that I kappa B alpha is rapidly resynthesized after loss, indicating that an autoregulatory mechanism is involved in the regulation of NF-kappa B function. We propose a mechanism for the activation of NF-kappa B through the modification and loss of I kappa B alpha, thereby establishing its role as a mediator of NF-kappa B activation.

Characterization of an immediate-early gene induced in adherent monocytes that encodes IκB-like activity

We have cloned a group of cDNAs representing mRNAs that are rapidly induced following adherence of human monocytes. One of the induced transcripts (MAD-3) encodes a protein of 317 amino acids with one domain containing five tandem repeats of the cdc10/ankyrin motif, which is 60% similar (46% identical) to the ankyrin repeat region of the precursor of NF-kappa B/KBF1 p50. The C-terminus has a putative protein kinase C phosphorylation site. In vitro translated MAD-3 protein was found to specifically inhibit the DNA-binding activity of the p50/p65 NF-kappa B complex but not that of the p50/p50 KBF1 factor or of other DNA-binding proteins. The MAD-3 cDNA encodes an I kappa B-like protein that is likely to be involved in regulation of transcriptional responses to NF-kappa B, including adhesion-dependent pathways of monocyte activation.

Influenza A Virus NS1 Protein Prevents Activation of NF-κB and Induction of Alpha/Beta Interferon

ABSTRACT The alpha/beta interferon (IFN-α/β) system represents one of the first lines of defense against virus infections. As a result, most viruses encode IFN antagonistic factors which enhance viral replication in their hosts. We have previously shown that a recombinant influenza A virus lacking the NS1 gene (delNS1) only replicates efficiently in IFN-α/β-deficient systems. Consistent with this observation, we found that infection of tissue culture cells with delNS1 virus, but not with wild-type influenza A virus, induced high levels of mRNA synthesis from IFN-α/β genes, including IFN-β. It is known that transactivation of the IFN-β promoter depends on NF-κB and several other transcription factors. Interestingly, cells infected with delNS1 virus showed high levels of NF-κB activation compared with those infected with wild-type virus. Expression of dominant-negative inhibitors of the NF-κB pathway during delNS1 virus infection prevented the transactivation of the IFN-β promoter, demonstrating a functional link between NF-κB activation and IFN-α/β synthesis in delNS1 virus-infected cells. Moreover, expression of the NS1 protein prevented virus- and/or double-stranded RNA (dsRNA)-mediated activation of the NF-κB pathway and of IFN-β synthesis. This inhibitory property of the NS1 protein of influenza A virus was dependent on its ability to bind dsRNA, supporting a model in which binding of NS1 to dsRNA generated during influenza virus infection prevents the activation of the IFN system. NS1-mediated inhibition of the NF-κB pathway may thus play a key role in the pathogenesis of influenza A virus.

An Essential Role of the NF-κB/Toll-Like Receptor Pathway in Induction of Inflammatory and Tissue-Repair Gene Expression by Necrotic Cells

Tissue damage induced by infection or injury can result in necrosis, a mode of cell death characterized by induction of an inflammatory response. In contrast, cells dying by apoptosis do not induce inflammation. However, the reasons for underlying differences between these two modes of cell death in inducing inflammation are not known. Here we show that necrotic cells, but not apoptotic cells, activate NF-κB and induce expression of genes involved in inflammatory and tissue-repair responses, including neutrophil-specific chemokine genes KC and macrophage-inflammatory protein-2, in viable fibroblasts and macrophages. Intriguingly, NF-κB activation by necrotic cells was dependent on Toll-like receptor 2, a signaling pathway that induces inflammation in response to microbial agents. These results have identified a novel mechanism by which cell necrosis, but not apoptosis, can induce expression of genes involved in inflammation and tissue-repair responses. Furthermore, these results also demonstrate that the NF-κB/Toll-like receptor 2 pathway can be activated both by exogenous microbial agents and endogenous inflammatory stimuli.

Oncogenic Ha-Ras-induced Signaling Activates NF-κB Transcriptional Activity, Which Is Required for Cellular Transformation

Ras proteins function in stimulating cell proliferation and differentiation through the activation of Raf-dependent and Raf-independent signal transduction pathways and the subsequent activation of specific transcription factors. The transcription factor NF-κB has been widely studied as a regulator of genes involved in immune and inflammatory responses. A variety of stimuli activate NF-κB through the induced phosphorylation and degradation of the inhibitor IκB followed by nuclear translocation of NF-κB. We show here that oncogenic forms of Ha-Ras activate NF-κB, not through induced nuclear translocation, but rather through the activation of the transcriptional function of the NF-κB RelA/p65 subunit. Importantly, RelA/p65 −/− cells are inefficient in the activation of κB-dependent gene expression in response to oncogenic Ras expression. Furthermore, IκBα expression blocks focus formation in NIH3T3 cells induced by oncogenic Ras. These results demonstrate that NF-κB is a critical downstream mediator of Ha-Ras signaling and oncogenic potential.

Dendritic Cell Development and Survival Require Distinct NF-κB Subunits

Abstract Despite the established role of dendritic cells (DCs) in regulating T lymphocyte activation, intracellular mechanisms responsible for controlling DC function are largely undefined. Here, we have studied DCs from mice deficient in the p50, RelA, and cRel subunits of the immunomodulatory NF-κB transcription factor. Although DC development and function was normal in mice lacking individual NF-κB subunits, development of doubly deficient p50 −/− RelA −/− DCs was significantly impaired. In contrast, DCs from p50 −/− cRel −/− mice developed normally, but CD40L- and TRANCE-induced survival and IL-12 production was abolished. Surprisingly, no significant impairment in MHC and costimulatory molecule expression was seen, despite significantly reduced κB site binding activity. These results therefore indicate essential, subunit-specific functions for NF-κB proteins in regulating DC development, survival, and cytokine production.

Interplay of IKK/NF-κB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder associated with dystrophin deficiency that results in chronic inflammation and severe skeletal muscle degeneration. In DMD mouse models and patients, we find that IkappaB kinase/NF-kappaB (IKK/NF-kappaB) signaling is persistently elevated in immune cells and regenerative muscle fibers. Ablation of 1 allele of the p65 subunit of NF-kappaB was sufficient to improve pathology in mdx mice, a model of DMD. In addition, conditional deletion of IKKbeta in mdx mice elucidated that NF-kappaB functions in activated macrophages to promote inflammation and muscle necrosis and in skeletal muscle fibers to limit regeneration through the inhibition of muscle progenitor cells. Furthermore, specific pharmacological inhibition of IKK resulted in improved pathology and muscle function in mdx mice. Collectively, these results underscore the critical role of NF-kappaB in the progression of muscular dystrophy and suggest the IKK/NF-kappaB signaling pathway as a potential therapeutic target for DMD.

Targeted Mutation of TNF Receptor I Rescues the RelA-Deficient Mouse and Reveals a Critical Role for NF-κB in Leukocyte Recruitment

NF-κB binding sites are present in the promoter regions of many acute phase and inflammatory response genes, suggesting that NF-κB plays an important role in the initiation of innate immune responses. However, targeted mutations of the various NF-κB family members have yet to identify members responsible for this critical role. RelA-deficient mice die on embryonic day 15 from TNF-α-induced liver degeneration. To investigate the importance of RelA in innate immunity, we genetically suppressed this embryonic lethality by breeding the RelA deficiency onto a TNFR type 1 (TNFR1)-deficient background. TNFR1/RelA-deficient mice were born healthy, but were susceptible to bacterial infections and bacteremia and died within a few weeks after birth. Hemopoiesis was intact in TNFR1/RelA-deficient newborns, but neutrophil emigration to alveoli during LPS-induced pneumonia was severely reduced relative to that in wild-type or TNFR1-deficient mice. In contrast, radiation chimeras reconstituted with RelA or TNFR1/RelA-deficient hemopoietic cells were healthy and demonstrated no defect in neutrophil emigration during LPS-induced pneumonia. Analysis of RNA harvested from the lungs of mice 4 h after LPS insufflation revealed that the induction of several genes important for neutrophil recruitment to the lung was significantly reduced in TNFR1/RelA-deficient mice relative to that in wild-type or TNFR1-deficient mice. These results suggest that TNFR1-independent activation of RelA is essential in cells of nonhemopoietic origin during the initiation of an innate immune response.

Degradation of Promoter-bound p65/RelA Is Essential for the Prompt Termination of the Nuclear Factor κB Response

Transcription factors of the nuclear factor (NF)-κB/Rel family translocate into the nucleus upon degradation of the IκBs. Postinduction repression of NF-κB activity depends on NF-κB–regulated resynthesis of IκBα, which dissociates NF-κB from DNA and exports it to the cytosol. We found that after activation, p65/RelA is degraded by the proteasome in the nucleus and in a DNA binding–dependent manner. If proteasome activity is blocked, NF-κB is not promptly removed from some target genes in spite of IκBα resynthesis and sustained transcription occurs. These results indicate that proteasomal degradation of p65/RelA does not merely regulate its stability and abundance, but also actively promotes transcriptional termination.

Mechanism of All-Trans Retinoic Acid Effect on Tumor-Associated Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSC) play an important role in tumor escape by suppressing T-cell responses. MDSC represent a group of cells of myeloid lineage at different stages of differentiation. Increased arginase activity and production of reactive oxygen species (ROS) are among the main functional characteristics of these cells. Recent studies have shown that all-trans retinoic acid (ATRA) had a potent activity in eliminating MDSC in cancer patients and in tumor-bearing mice. ATRA differentiates these cells into mature myeloid cells. However, the mechanism of this effect is unclear. Here, we have shown that ATRA dramatically and specifically up-regulated gene expression and protein level of glutathione synthase (GSS) in MDSC. This resulted in accumulation of glutathione (GSH) in these cells, observed in both mice and cancer patients. Blockade of GSH synthesis cancelled the effect of ATRA on MDSC. Accumulation of GSH in these cells using N-acetyl-L-cysteine mimicked the effect of ATRA on MDSC differentiation. Analysis of potential mechanisms of ATRA effect on GSS revealed that ATRA regulates its expression not by directly binding to the promoter but primarily via activation of extracellular signal-regulated kinase 1/2. Thus, ATRA induced differentiation of MDSC primarily via neutralization of high ROS production in these cells. This novel mechanism involves specific up-regulation of GSS and accumulation of GSH and could be used in developing and monitoring therapeutic application of ATRA.