Toshifumi Tetsuka

The role of p38 mitogen‐activated protein kinase in IL‐6 and IL‐8 production from the TNF‐α‐ or IL‐1β‐stimulated rheumatoid synovial fibroblasts

We examined the role of p38 mitogen‐activated protein (MAP) kinase in the tumor necrosis factor α (TNF‐α)‐ or interleukin‐1β (IL‐1β)‐induced production of interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8) in fresh rheumatoid synovial fibroblast (RSF) cultures concomitantly with the induction of p38 MAP kinase activity. Pretreatment of RSF with a specific p38 MAP kinase inhibitor, SB203580, blocked the induction of IL‐6 and IL‐8 without affecting nuclear translocation of nuclear factor κB (NF‐κB) or IL‐6 and IL‐8 mRNA levels. These findings suggest that p38 MAP kinase inhibitor may have synergistic, rather than additive, effect for the treatment of rheumatoid arthritis.

Antioxidants modulate acute solar ultraviolet radiation-induced NF-kappa-B activation in a human keratinocyte cell line

Exposure of the human skin to ultraviolet radiation (UVR) leads to depletion of cutaneous antioxidants, regulation of gene expression and ultimately to the development of skin diseases. Although exogenous supplementation of antioxidants prevents UVR-induced photooxidative damage, their effects on components of cell signalling pathways leading to gene expression has not been clearly established. In the present study, the effects of the antioxidants alpha-lipoic acid, N-acetyl-L-cysteine (NAC) and the flavonoid extract silymarin were investigated for their ability to modulate the activation of the transcription factors nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) in HaCaT keratinocytes after exposure to a solar UV simulator. The activation of NF-kappaB and AP-1 showed a similar temporal pattern: activation was detected 2 h after UV exposure and maintained for up to 8 h. To determine the capacity of activated NF-kappaB to stimulate transcription, NF-kappaB-dependent gene expression was measured using a reporter gene assay. The effects of the antioxidants on NF-kappaB and AP-1 activation were evaluated 3 h after exposure. While a high concentration of NAC could achieve a complete inhibition, low concentrations of alpha-lipoic acid and silymarin were shown to significantly inhibit NF-kappaB activation. In contrast, AP-1 activation was only partially inhibited by NAC, and not at all by alpha-lipoic acid or silymarin. These results indicate that antioxidants such as alpha-lipoic acid and silymarin can efficiently modulate the cellular response to UVR through their selective action on NF-kappaB activation.

Inhibition of nuclear factor-κB-mediated transcription by association with the amino-terminal enhancer of split, a groucho-related protein lacking WD40 repeats.

The amino-terminal enhancer of split (AES) encodes a 197-amino acid protein that is homologous to the NH2-terminal domain of the DrosophilaGroucho protein but lacks COOH-terminal WD40 repeats. Although theDrosophila Groucho protein and its mammalian homologs, transducin-like enhancer of split proteins, are known to act as non-DNA binding corepressors, the role of the AES protein remains unclarified. Using the yeast two-hybrid system, we have identified the protein-protein interaction between AES and the p65 (RelA) subunit of the transcription factor nuclear factor κB (NF-κB), which activates various target genes involved in inflammation, apoptosis, and embryonic development. The interaction between AES and p65 was confirmed byin vitro glutathione S-transferase pull down assay and by in vivo co-immunoprecipitation study. In transient transfection assays, AES repressed p65-driven gene expression. AES also inhibited NF-κB-dependent gene expression induced by tumor necrosis factor α, interleukin-1β, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1, which is an upstream kinase for NF-κB activation. These data indicate that AES acts as a corepressor for NF-κB and suggest that AES may play a pivotal role in the regulation of NF-κB target genes.

Induction of Notch signaling by tumor necrosis factor in rheumatoid synovial fibroblasts.

Rheumatoid arthritis (RA) is characterized by progressive inflammation associated with abberrant proliferation of synoviocytes. In order to explore the characteristics of rheumatoid synovial fibroblasts (RSF), we performed the comparative gene expression profile analysis between RSF and normal synovial fibroblasts (NSF) upon tumor necrosis factor (TNF) stimulation. As an initial screening for the genes preferentially induced by TNF in RSF compared with NSF, we have adopted a cDNA array containing well-defined sets of genes responsible for cell growth, cell fate determination, and cellular invasiveness. Differentially expressed genes of interest were confirmed using real-time RT–PCR. We found that TNF induced the expression of Notch-1, Notch-4, and Jagged-2 in RSF. The expression of these proteins was detected in the RA synovial tissues. The nucleus of RA synoviocytes showed strong staining with anti-Notch-1 and Notch-4 antibody. TNF induced the nuclear translocation of Notch intracellular domain in RSF, indicating the elicitation of the Notch signaling. Notch-1, Notch-4, and Jagged-2 proteins were also detected in the developing synovium of mouse embryo. Thus, RSF may have re-acquired the primordial phenotype, accounting for the hyperproliferation and aggressive invasiveness, exhibiting tumor-like phenotype.

NF-κB p65 transactivation domain is involved in the NF-κB-inducing kinase pathway☆☆☆

Abstract NF-κB-inducing kinase (NIK) is involved in the signal transduction pathway leading to the NF-κB activation. In this report, we demonstrate that the NIK-mediated NF-κB activation involves the transactivation (TA) domain of p65 subunit of NF-κB and the nuclear translocation of IKKα. By using luciferase assay, we found that both IKKα and IKKβ could activate NF-κB in synergy with NIK. Interestingly, although IKKβ stimulated the NIK-mediated IκB degradation, IKKα stimulated the action of NF-κB without enhancing the IκB degradation. By using heterologous transactivation system with Gal4 DNA-binding domain in fusion with various portions of p65 TA domain, we found that the transactivation domain 1 (TA1) of p65 serves as the direct target for the NIK–IKKα cascade and that the serine residue at 536 within p65 TA1 is indispensable for this action. Furthermore, we found that this action of NIK depends on the energy-dependent action of Ras-related protein (Ran) since the dominant negative mutant of Ran (RanQ69L) inhibited the transcriptional activity of p65 by preventing the nuclear import of IKKα.

RelA-Associated Inhibitor Blocks Transcription of Human Immunodeficiency Virus Type 1 by Inhibiting NF-κB and Sp1 Actions

ABSTRACT RelA-associated inhibitor (RAI) is an inhibitor of nuclear factor κB (NF-κB) newly identified by yeast two-hybrid screen as an interacting protein of the p65 (RelA) subunit. In this study, we attempted to examine the effect of RAI on transcription and replication of human immunodeficiency virus type 1 (HIV-1). We found that RAI inhibited gene expression from the HIV-1 long terminal repeat (LTR) even at the basal level. Upon in vitro DNA-binding reactions, RAI could directly block the DNA-binding of p65 subunit of NF-κB but not that of the p50 subunit or AP1. We found that RAI could also inhibit the DNA-binding of Sp1 and thus inhibit the basal HIV-1 promoter activity. We further examined the effects of RAI on Sp1 and found that RAI colocalizes with Sp1 in the nucleus and interacts with Sp1 in vitro and in vivo. Moreover, we found that RAI efficiently blocked the HIV-1 replication when cotransfected with a full-length HIV-1 clone. These findings indicate that RAI acts as an efficient inhibitor of HIV-1 gene expression in which both NF-κB and Sp1 play major roles.

Evidence that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-κB

The transcription factor NF-κB is a positive transcription factor for a number of genes and has been recognized as an anti-apoptotic regulator. However, the mechanism by which NF-κB blocks apoptosis is still controversial. Here, we demonstrate the evidence that NF-κB could attenuate the TNF-α-induced apoptosis without de novo protein synthesis using human pancreatic cancer cell lines, MIA PaCa-2 and Capan-2. The TNF-α-induced apoptosis was blocked by IL-1β, a potent inducer of NF-κB activation. This inhibitory effect of IL-1β was evident when cells were treated with protein synthesis inhibitors such as cycloheximide (CHX). Moreover, NF-κB decoy oligonucleotides could not block the anti-apoptotic effect of IL-1β at doses sufficient to block the NF-κB-dependent transcription induced by IL-1β. To confirm the role of NF-κB in blocking apoptosis, we generated stable cell lines expressing IκBΔN, a highly stable form of IκBα, a cytoplasmic inhibitor of NF-κB. In these stable transfectants, the anti-apoptotic effect of IL-1β was totally abolished, indicating that the anti-apoptotic action of IL-1β could be ascribed to the NF-κB action. These findings show that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-κB and support the possibility that NF-κB could exert its anti-apoptotic action through protein-protein interaction.

3-Methylcholanthrene Activates Human Immunodeficiency Virus Type 1 Replication via Aryl Hydrocarbon Receptor

We found that 3‐methylcholanthrene (3‐MC) could induce the reactivation of human immunodeficiency virus type 1 (HIV‐1) replication in OM 10.1 cell, promyelocytic cell line latently infected with HIV‐1. Transient luciferase expression experiments have revealed no particular transcription factors that are responsible for the effect of 3‐MC in inducing HIV‐1 gene expression as HIV‐1 LTR mutants lacking various upstream transcriptional activators similarly responded to 3‐MC. In addition, there was no effect of 3‐MC on the DNA binding activity of nuclear factor‐kappa B (NF‐κB) that was previously reported to be crucial for the effect of 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD), a chemical homologue of 3‐MC. However, overexpression of wild type aryl hydrocarbon receptor (AhR), a nuclear receptor of polycyclic aromatic hydrocarbons (PAHs) such as 3‐MC, augmented the effect of 3‐MC in the induction of gene expression from HIV‐1 LTR. Moreover, a dominant negative mutant of AhR dramatically reduced the 3‐MC‐mediated activation of HIV‐1 LTR. These findings suggest that 3‐MC stimulates HIV‐1 transcription by interacting with general transcription factors. Our observations indicate that chronic exposure of the HIV‐1 infected individuals to PAHs may be contributable to the clinical development of acquired immunodeficiency syndrome (AIDS) among the individuals infected with HIV.

Assignment of the IκB-β gene NFKBIB to human chromosome band 19q13.1 by in situ hybridization

We have isolated the full-length NFKBIB cDNA clone from a human placenta Matchmaker cDNA library (Clontech, Palo Alto CA) by two-hybrid screening using yeast (Iwabuchi et al., 1994). The NFKBIB gene (alias IKBB, TRIP9) encodes the IÎB-ß protein, an inhibitory molecule of transcription factor NF-ÎB (reviewed by Baeuerle and Baichwal, 1997; Okamoto et al., 1997; Baldwin, 1996; Beg and Baldwin, 1993). The IÎB proteins comprise a distinct family including the following members: IÎB-· and IÎB-ß, which are cytoplasmic in location and inhibit the DNA binding of NF-ÎB (Ghosh and Baltimore, 1990; Link et al., 1992); the product of the bcl-3 proto-oncogene (Nolan et al., 1993), which is nuclear in location and inhibits the DNA-binding of NF-ÎB but, paradoxically, activates transcription when interacting with a homodimer complex of p50, one of the subunits of NF-ÎB (Bours et al., 1993; Fujita et al., 1993); and the proteins corresponding to the C-terminal portions of p105 and p100, which are synthesized by alternative splicing of the respective genes and act as IÎB molecules, thus named IÎB-Á and IÎB-‰, respectively (Inoue et al., 1992; Dobrzanski et al., 1995). It is noted that the human IÎB-ß gene was initially identified as a transcriptional co-activator for thyroid hormone receptor and thus named TRIP9 (Lee et al., 1995). Since NF-ÎB regulates a large number of genes that positively control various aspects of the immune and inflammatory response, IÎB proteins are considered to be crucial regulators of these biological responses. Moreover, recent studies have indicated that NF-ÎB blocks the apoptosis pathway (Beg and Baltimore, 1996; Wu et al., 1996; Wang et al., 1996), thus also highlighting the regulatory role of IÎB proteins. We have localized the NFKBIB gene to chromosome 19 band q13.1. The genetic loci did not reveal any association with a hereditary disease whose causative gene has not been identified. However, we found that NFKBIB mapped in the close vicinity of bcl-3 located at 19q13.2 (McKeithan et al., 1990). It is likely that these two genes share the same ancestral origin and might have been generated by localized gene duplication.

Role of thioredoxin in the redox regulation of gene expression in inflammatory diseases

Oxygen is an essential molecule to generate energy for all aerobic life forms including humans. However, while oxygen is indispensable for such cells to obtain the essential chemical energy as a form of adenosine triphosphate (ATP), which is called respiration, incomplete reduction of oxygen generates highly active, as well as toxic, forms called radical oxygen intermediates (ROI). In addition to being produced as physiological side-products of electron transfer reactions using oxygen as an electron acceptor, ROI are produced in the cell by various environmental stimuli such as infection of microbes (viruses, bacteria, etc.), ionizing and UV irradiation, and pollutants (i.e. oxidants), which are collectively called “oxidative stress”. These ROI are highly reactive with biological macromolecules, producing lipid peroxides (which are often radicals), inactivating proteins and mutating DNA (by producing 8-hydroxy-deoxy-guanosine, 8-OH-dG, or breaking nucleic acid chains).