Transient Nuclear Factor κB (NF-κB) Activation Stimulated by Interleukin-1β May Be Partly Dependent on Proteasome Activity, but Not Phosphorylation and Ubiquitination of the IκBα Molecule, in C6 Glioma Cells

Abstract

We previously reported that several stresses can induce cytokine-induced neutrophil chemoattractant expression in a nuclear factor B (NF-B)-dependent manner. In this study, we focused further on the regulation of NF-B. The activation of NF-B and the subsequent cytokine-induced neutrophil chemoattractant induction in response to interleukin-1␤ (IL-1␤) were inhibited by proteasome inhibitors, MG132 and proteasome inhibitor I. Translocation of NF-B into nuclei occurs by the phosphorylation, multi-ubiquitination, and degradation of IB␣, a regulatory protein of NF-B. Nascent IB␣ began to degrade 5 min after treatment with IL-1␤ and disappeared completely after 15 min. However, IB␣ returned to basal levels after 45-60 min. Interestingly, resynthesized IB␣ was already phosphorylated at Ser-32. These results suggest that 1) the upstream signals are still activated, although the translocation of NF-B peaks at 15 min; and 2) the regulated protein(s) acts downstream of IB␣ phosphorylation. Western blotting showed that the resynthesized and phosphorylated IB molecules were also upward-shifted by multi-ubiquitination in response to IL-1␤ treatment. On the other hand, ATP-dependent Leu-Leu-Val-Tyr cleaving activity transiently increased, peaked at 15 min, and then decreased to basal levels at 60 min. Furthermore, the cytosolic fraction that was stimulated by IL-1␤ for 15 min, but not for 0 and 60 min, could degrade phosphorylated and multi-ubiquitinated IB␣. These results indicate that the transient translocation of NF-B in response to IL-1␤ may be partly dependent on transient proteasome activation. Nuclear factor B (NF-B)1 participates in the regulation of the expression of multiple immediate-early genes involved in immune, acute-phase, and inflammatory responses (1). NF-B is a heterodimer protein of the Rel family of transcription factors. In mammalian cells, the factors include p65 (RelA), RelB, c-Rel, p50/p105 (NF-B1), and p52/p100 (NF-B2). NF-B proteins are constitutively present in cells and are retained in the cytoplasm associated with the inhibitory protein IB (2, 3). Activated NF-B complexes, typically composed of p50 and p65, are translocated to the nucleus in response to several cytokines (TNF-␣, IL-1␤, and IL-2), bacterial endotoxin, and stresses (UV, H 2 O 2 ).(1, 4 -6). The activation of NF-B appears to require the phosphorylation and degradation of the IB proteins, thereby allowing the rapid translocation of NF-B from the cytoplasm to the nucleus (4, 7-9). In particular, it has been shown that the phosphorylation of Ser-32 and Ser-36 and the ubiquitination at Lys-21 and Lys-22 are essential for targeting IB for signal-induced degradation by the ubiquitin/proteasome system (10). The ubiquitin-dependent degradation of regulatory shortlived proteins plays an important role in cellular processes, including the cell cycle, immune system functions, inflammatory responses, and tissue differentiation. A key element in the regulation process is E3, a member of the ubiquitin-substrate ligase family of enzymes. After bin...

IL-1 regulates in vivo C—X—C chemokine induction and neutrophil sequestration following endotoxemia

“…The transcription of multiple chemokine genes, including IL-8, CINC-1 and MIP-2, are regulated by the transcription factors nuclear factor kappa B (NF-kB) and activating protein-1 (AP-1). [35][36][37][38] Thus IL-1Ra may inhibit CINC-1 and MIP-2 gene expression via inhibition of IL-1-induced activation of NF-kB or AP-1. Alternatively, IL-1 is capable of stabilization of chemokine mRNA with an increase in mRNA halflife.…”

Correlation between Delayed Neuronal Cell Death and Selective Decrease in Phosphatidylinositol 4-Kinase Expression in the CA1 Subfield of the Hippocampus after Transient Forebrain Ischemia

“…When the cells became subconfluent, they were cultured in a mixture of 5% carbon dioxide and the balance of nitrogen in a humidified incubator (ANX-1, HIRASAWA, Tokyo, Japan) at 37°C within a sealed, anaerobic, gloved cabinet containing a catalyst to scavenge free oxygen as described previously (Ko et al, 2002;Tanaka et al, 2000;Uehara et al, 1999a). The medium was then changed, and the cells were cultured under normal conditions for given periods (reoxygenation).…”

“…An aliquot (2 L) of RT product was mixed with 2 U AmpliTaq polymerase and a 0.2-mol/L concentration of each of the following sense and antisense primers in a final volume of 20 L (Uehara et al, 1999a): rat PI 4-K, 5Ј-ATC CAG ATC GCT TCC AGG TAA-3Ј (positions 2,934 to 2,954; upstream) and 5Ј-AAC GGC ACT TCC ACT CCA TTC-3Ј (positions 3,931 to 3,951; downstream); rat PI 3-K (p85), 5Ј-TGT TGC CCC GCC TCT CCT TAT-3Ј (positions 378 to 398; upstream) and 5Ј-ATT TCC TCC TTT CCT TAG TGT-3Ј (positions 1,111 to 1,131; downstream); and rat GAPDH, 5Ј-AAA CCC ATC ACC ATC TTC CAG-3Ј (positions 238 to 258; upstream) and 5Ј-AGG GGC CAT CCA CAG TCT CAG-3Ј (positions 578 to 598; downstream).…”

Herbimycin A Abrogates Nuclear Factor-κB Activation by Interacting Preferentially with the IκB Kinase β Subunit

“…We have demonstrated previously that treatment with herbimycin A selectively attenuates the gene induction regulated by NF-B in glial cells (Nishiya et al, 1995(Nishiya et al, , 2000Uehara et al, 1998Uehara et al, , 1999. In this report, we attempted to elucidate the possible target for herbimycin A.…”

“…We have reported previously that herbimycin A, a potent Src tyrosine kinase inhibitor, suppresses the NF-B activation and subsequent induction of inducible nitric oxide synthase and chemokines by treatment with several stimuli in C6 glioma cells (Uehara et al, 1989(Uehara et al, , 1998(Uehara et al, , 1999Nishiya et al, 1995Nishiya et al, , 2000. However, the target molecule of herbimycin A in NF-B-activating pathways remains to be identified.…”