Hakju Kwon

Interferon regulatory factors: the next generation

Interferons are a large family of multifunctional secreted proteins involved in antiviral defense, cell growth regulation and immune activation. Viral infection induces transcription of multiple IFN genes, a response that is in part mediated by the interferon regulatory factors (IRFs). The initially characterized members IRF-1 and IRF-2 are now part of a growing family of transcriptional regulators that has expanded to nine members. The functions of the IRFs have also expanded to include distinct roles in biological processes such as pathogen response, cytokine signaling, cell growth regulation and hematopoietic development. The aim of this review is to provide an update on the novel discoveries in the area of IRF transcription factors and the important roles of the new generation of IRFs--particularly IRF-3, IRF-4 and IRF-7.

Hostile takeovers: viral appropriation of the NF-kB pathway

Transcriptional regulators of the NF-kB/IkB family promote the expression of well over 100 target genes, the majority of which participate in the host immune response (1). These proteins include a multitude of cytokines and chemokines, receptors required for immune recognition, proteins involved in antigen presentation, and adhesion receptors involved in transmigration across blood vessels walls. Because of this extensive role in immune action, NF-kB has been termed the central mediator of the immune response. Gene knockout and other studies establish roles for NF-kB in the ontogeny of the immune system but also demonstrate that NF-kB participates at multiple steps during oncogenesis (2) and the regulation of programmed cell death (3).

I kappaB alpha physically interacts with a cytoskeleton-associated protein through its signal response domain.

The I kappaB alpha protein is a key molecular target involved in the control of NF-kappaB/Rel transcription factors during viral infection or inflammatory reactions. This NF-kappaB-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets I kappaB alpha for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the I kappaB alpha inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of I kappaB alpha as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length I kappaB alpha protein but not with N-terminal-deletion-containing versions of I kappaB alpha. I kappaB alpha interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of I kappaB alpha. Importantly, in HeLa and 293 cells, endogenous and transfected I kappaB alpha coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and I kappaB alpha colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and I kappaB alpha were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, I kappaB alpha colocalized with alpha-tubulin filaments. Taken together, these results highlight an intriguing interaction between the I kappaB alpha protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation.

Disruption of NF-κB Signaling Reveals a Novel Role for NF-κB in the Regulation of TNF-Related Apoptosis-Inducing Ligand Expression

The NF-κB family of transcription factors functions broadly in the host control of immunoregulatory gene expression, inflammation, and apoptosis. Using Jurkat T cells engineered to inducibly express a transdominant repressor of IκBα, we examined the role of NF-κB in the regulation of cytokine and apoptotic gene expression. In this T cell model, as well as in primary T lymphocytes, expression of TNF-related apoptosis-inducing ligand (TRAIL) apoptotic signaling protein was dramatically down-regulated by inhibition of NF-κB binding activity. TRAIL acts through membrane death receptors to induce apoptosis of activated T lymphocytes and can be up-regulated by a variety of physiological and pharmacological inducers. However, regulation of TRAIL gene expression has not been defined. Treatment with TCR mimetics (PMA/ionomycin, PHA, and anti-CD3/CD28 Abs) resulted in a rapid increase in the expression of TRAIL mRNA and cell surface TRAIL protein. Induction of the transdominant repressor of IκBα dramatically down-regulated surface expression of TRAIL, indicating an essential role for NF-κB in the regulation of TRAIL. The induced expression of TRAIL was linked to a c-Rel binding site in the proximal TRAIL promoter at position −256 to −265; mutation of this site or an adjacent κB site resulted in a complete loss of the inducibility of the TRAIL promoter. The regulation of TRAIL expression by NF-κB may represent a general mechanism that contributes to the control of TRAIL-mediated apoptosis in T lymphocytes.

NF-κB/Rel Regulates Inhibitory and Excitatory Neuronal Function and Synaptic Plasticity

ABSTRACT Changes in synaptic plasticity required for memory formation are dynamically regulated through opposing excitatory and inhibitory neurotransmissions. To explore the potential contribution of NF-κB/Rel to these processes, we generated transgenic mice conditionally expressing a potent NF-κB/Rel inhibitor termed IκBα superrepressor (IκBα-SR). Using the prion promoter-enhancer, IκBα-SR is robustly expressed in inhibitory GABAergic interneurons and, at lower levels, in excitatory neurons but not in glia. This neuronal pattern of IκBα-SR expression leads to decreased expression of glutamate decarboxylase 65 (GAD65), the enzyme required for synthesis of the major inhibitory neurotransmitter, γ-aminobutyric acid (GABA) in GABAergic interneurons. IκBα-SR expression also results in diminished basal GluR1 levels and impaired synaptic strength (input/output function), both of which are fully restored following activity-based task learning. Consistent with diminished GAD65-derived inhibitory tone and enhanced excitatory firing, IκBα-SR+ mice exhibit increased late-phase long-term potentiation, hyperactivity, seizures, increased exploratory activity, and enhanced spatial learning and memory. IκBα-SR+ neurons also express higher levels of the activity-regulated, cytoskeleton-associated (Arc) protein, consistent with neuronal hyperexcitability. These findings suggest that NF-κB/Rel transcription factors act as pivotal regulators of activity-dependent inhibitory and excitatory neuronal function regulating synaptic plasticity and memory.

Activation of the IκBα kinase (IKK) complex by double-stranded RNA-binding defective and catalytic inactive mutants of the interferon-inducible protein kinase PKR

The interferon (IFN)-inducible double stranded (ds) RNA-activated protein kinase PKR plays an important role in protein synthesis by modulating the phosphorylation of the α-subunit of eukaryotic initiation fact 2 (eIF-2α). In addition to translational control, PKR has been implicated in several signaling pathways leading to gene transcription. For example, PKR induces IκBα kinase (IKK) activity and IκBα phosphorylation leading to the induction of NF-κB-mediated gene transcription. Recent findings suggested that NF-κB activation by PKR does not require the catalytic activity of the kinase. Here, we provide novel evidence that induction of IKK and NF-κB activities proceeds independently of the dsRNA-binding properties of PKR and also verify the kinase-free role of PKR in this process. We also show that the effects of PKR mutants on IKK and NF-κB activation are independent of cell transformation but are dependent on the amount of the mutant PKR proteins expressed in cells. These data strongly support an indirect role of PKR in IκBα phosphorylation by modulating IKK activity through pathways that do not utilize the enzymatic and dsRNA-binding properties of PKR.

Identification by In Vivo Genomic Footprinting of a Transcriptional Switch Containing NF-κB and Sp1 That Regulates the IκBα Promoter

ABSTRACT In unstimulated cells, NF-κB transcription factors are retained in the cytoplasm by inhibitory IκB proteins. Upon stimulation by multiple inducers including cytokines or viruses, IκBα is rapidly phosphorylated and degraded, resulting in the release of NF-κB and the subsequent increase in NF-κB-regulated gene expression. IκBα gene expression is also regulated by an NF-κB autoregulatory mechanism, via NF-κB binding sites in the IκBα promoter. In previous studies, tetracycline-inducible expression of transdominant repressors of IκBα (TD-IκBα) progressively decreased endogenous IκBα protein levels. In the present study, we demonstrate that expression of TD-IκBα blocked phorbol myristate acetate-phytohemagglutinin or tumor necrosis factor alpha-induced IκBα gene transcription and abolished NF-κB DNA binding activity, due to the continued cytoplasmic sequestration of RelA(p65) by TD-IκBα. In vivo genomic footprinting revealed stimulus-responsive protein-DNA binding not only to the −63 to −53 κB1 site but also to the adjacent −44 to −36 Sp1 site of the IκBα promoter. In vivo protection of both sites was inhibited by tetracycline-inducible TD-IκBα expression. Prolonged NF-κB binding and a temporal switch in the composition of NF-κB complexes bound to the −63 to −53 κB1 site of the IκBα promoter were also observed; with time after induction, decreased levels of transcriptionally active p50-p65 and increased p50–c-Rel heterodimers were detected at the κB1 site. Mutation of either the κB1 site or the Sp1 site abolished transcription factor binding to the respective sites and the inducibility of the IκBα promoter in transient transfection studies. These observations provide the first in vivo characterization of a promoter proximal transcriptional switch involving NF-κB and Sp1 that is essential for autoregulation of the IκBα promoter.

Taxol selectively blocks microtubule dependent NF-κB activation by phorbol ester via inhibition of IκBα phosphorylation and degradation

Activation of the NF-κB transcription factors has been shown to be directly influenced by changes in the microtubule cytoskeleton network. To better understand cytoskeletal regulation of NF-κB, experiments were performed to determine whether the microtubule (MT) stabilizing agent taxol could modulate NF-κB activation in the presence of different NF-κB inducers. Pretreatment of murine NIH3T3 and human 293 cells with 5 μM taxol resulted in complete inhibition of phorbol, 12-myristate, 13-acetate (PMA) mediated NF-κB activation, detected as the loss of DNA binding and reduced NF-κB dependent reporter gene activity. Furthermore, in COS-7 and NIH3T3 cells, PMA-induced IκBα turnover was dramatically reduced in taxol treated cells, mediated via the inhibition of IκBα phosphorylation. However, taxol did not prevent TNF-α induced IκBα phosphorylation, degradation, or NF-κB activation, indicating that TNF-α acts through a microtubule-independent pathway. In vitro kinase assays with PMA stimulated cell extracts demonstrated that taxol reduced protein kinase C activity by 30%, thus implicating the loss of PKC activity as a possible regulatory target of taxol-mediated suppression of NF-κB. Since PMA causes modulation of cytoarchitecture through PKC activation, microtubule integrity and cell morphology was analysed by indirect immunofluorescence. Both PMA and nocodazole, a MT depolymerizing agent, caused microtubule depolymerization, whereas TNF-α did not alter MT integrity; concomitant taxol treatment blocked both nocodazole and PMA induced depolymerization of MTs, as well as NF-κB induction, thus demonstrating a link between microtubule depolymerization and NF-κB activation. These observations illustrate a novel biological activity of taxol as a selective inhibitor of NF-κB activity, suggesting a link between the state of microtubule integrity and gene regulation.

NF-κB activation and HIV-1 induced apoptosis

Abstract HIV infection leads to the progressive loss of CD4 + T cells and the near complete destruction of the immune system in the majority of infected individuals. High levels of viral gene expression and replication result in part from the activation of NF-κB transcription factors, which in addition to orchestrating the host inflammatory response also activate the HIV-1 long terminal repeat. NF-κB induces the expression of numerous cytokine, chemokine, growth factor and immunoregulatory genes, many of which promote HIV-1 replication. Thus, NF-κB activation represents a double edged sword in HIV-1 infected cells, since stimuli that induce an NF-κB mediated immune response will also lead to enhanced HIV-1 transcription. NF-κB has also been implicated in apoptotic signaling, protecting cells from programmed cell death under most circumstances and accelerating apoptosis in others. Therefore, activation of NF-κB can impact upon HIV-1 replication and pathogenesis at many levels, making the relationship between HIV-1 expression and NF-κB activation multi-faceted. This review will attempt to analyse the many faces and functions of NF-κB in the HIV-1 lifecycle.

Cellular and viral protein interactions regulating I kappa B alpha activity during human retrovirus infection.

NF‐κB/Rel transcription factors participate in the activation of numerous genes involved in immune regulation/inflammation including cytokines, cell surface receptors, adhesion molecules, and acute phase proteins. NF‐κB activity is controlled by inhibitory proteins, IκBs, that maintain the DNA‐binding forms of NF‐κB in an inactive state in the cytoplasm. Many viruses, including the human retroviruses HIV‐1 and HTLV‐1, also utilize the NF‐κB/IκB pathway to their transcriptional advantage during viral infection. Our recent studies have focused on the IκBα inhibitor and have characterized several protein interactions that modulate the functional activity of IκBα during human retrovirus infection. In this article, we summarize recent studies demonstrating that (1) chronic HIV‐1 infection of human myelomonoblastic PLB‐985 cells leads to constitutive NF‐κB activity, activated in part due to enhanced IκBα turnover and increased NF‐κB/Rel production; (2) HTLV‐1 Tax protein physically associates with the IκBα protein in vivo and in vitro and also mediates a 20‐ to 40‐fold stimulation of NF‐κB DNA binding activity mediated via an enhancement of NF‐κB dimer formation; (3) casein kinase II phosphorylates IκBα at multiple sites in the C‐terminal PEST domain and regulates IκBα function; (4) transdominant forms of IκBα, mutated in critical Ser or Thr residues required for inducer‐mediated (S32A,S36A) and/or constitutive phosphorylation block HIV LTR trans‐activation and also effectively inhibit HIV‐1 multiplication in a single cycle infection model; and (5) the amino‐terminal 55aa of IκBα (NIK) interacts with the human homologue of dynein light chain 1, a small 9‐kDa human homologue of the dynein light chain protein involved in microtubule and cytoskeletal dynamics. Together, our results highlight a number of intriguing molecular interactions between IκBα and cellular or viral proteins that modulate transcription factor activity and nuclear‐cytoplasmic flow of host proteins. J. Leukoc. Biol. 62: 82–92; 1997.