Rapid Up-regulation of IκBβ and Abrogation of NF-κB Activity in Peritoneal Macrophages Stimulated with Lipopolysaccharide

Abstract

Lipopolysaccharide (LPS) administration to mice elicited the activation of nuclear factor B (NF-B) in several tissues including liver and macrophages. Maximal activation was observed 1 h after treatment but declined at 3 and 6 h. The levels of IB␣ and IB␤ were analyzed during this period in an attempt to correlate NF-B activity with IB resynthesis. Degradation of IB␣ was very rapid and was followed by recovery 1 h after LPS administration. IB␤ degradation, which has been associated with persistent NF-B activation, was complete at 1 h. However, a rapid recovery of IB␤ in these tissues was observed at 3 h in parallel with the abrogation of NF-B activity. Immunolocalization of newly synthesized IB␤ by confocal microscopy revealed its preferential accumulation in the cytosol. Analysis of IB␤ by Western blot using high resolution polyacrylamide gel electrophoresis showed the presence of two bands in cytosolic extracts of LPS-treated macrophages at 3 h, but only one band with the same mobility as the control was detected at 6 h. Moreover, treatment of extracts of resynthesized IB␤ with alkaline phosphatase resulted in the accumulation of the protein of slightly higher electrophoretic mobility, indicating the prevalence of a rapid phosphorylation of the newly synthesized IB␤. At the mRNA level, up-regulation of IB␤ was observed in macrophages stimulated for 1 h with LPS. When the effect of pro-inflammatory cytokines was investigated, tumor necrosis factor ␣, but not interleukin-1 or interferon-␥, promoted an important degradation of IB␤ followed by an increase in the mRNA at 1 h. These results suggest the existence of LPS-and tumor necrosis factor ␣-specific pathways involved in a rapid IB␤ degradation and resynthesis and might explain the transient period of activation of NF-B in these tissues upon stimulation with these factors. This rapid control of NF-B function may contribute to the attenuation of the inflammatory response of these cells. Nuclear factor B (NF-B)1 participates in the regulation of the expression of multiple immediate early genes involved in the immune, acute phase, and inflammatory responses (1). NF-B is a heterodimer of proteins of the Rel family of transcription factors. In mammalian cells, they include p65 (Rel A), Rel B, the proto-oncogene c-Rel, p50/p105 (NF-B1), and p52/ p100 (NF-B2) (1, 2). NF-B proteins are constitutively present in the cell, but they are retained in the cytoplasm associated with inhibitory proteins known as IB (3, 4). Activated NF⅐B complexes, typically composed of p50 and p65, are translocated to the nucleus in response to mitogens, cytokines (IL-1␤, IL-2, and TNF-␣), and bacterial lipopolysaccharide and lipopeptides (1, 5-8). Activation of NF-B appears to require phosphorylation and degradation of the IB proteins, thereby allowing the rapid translocation of NF-B from the cytoplasm to the nucleus (7, 9 -11). Several IB proteins have been characterized including IB␣, IB␤, IB␥, and the candidate oncogene 4,12). All these proteins share a characteristic ankyrin repeat motif,...

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