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

We previously reported that several stresses can induce cytokine- induced neutrophil chemoattractant expression in a nuclear factor κB (NF- κB)-dependent manner. In this study, we focused further on the regulation of NF-κB. The activation of NF-κB and the subsequent cytokine-induced neutrophil chemoattractant induction in response to interleukin-1β (IL-1β were inhibited by proteasome inhibitors, MG132 and proteasome inhibitor I. Translocation of NF-κB into nuclei occurs by the phosphorylation, multi- ubiquitination, and degradation of IκBα, a regulatory protein of NF-κB. Nascent IκBα began to degrade 5 min after treatment with IL-1β and disappeared completely after 15 min. However, IκBα returned to basal levels after 45-60 min. Interestingly, resynthesized IκBα was already phosphorylated at Ser32. These results suggest that 1) the upstream signals are still activated, although the translocation of NF-κB peaks at 15 min; and 2) the regulated protein(s) acts downstream of IκBα phosphorylation. Western blotting showed that the resynthesized and phosphorylated IκB molecules were also upward-shifted by multi-ubiquitination in response to IL- 1β 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-1β for 15 min, but not for 0 and 60 min, could degrade phosphorylated and multi-ubiquitinated IκBα. These results indicate that the transient translocation of NF-κB in response to IL-1β may be partly dependent on transient proteasome activation.