Low nuclear levels of nuclear factor-?B are essential for KC self-induction in astrocytes: Requirements for shuttling and phosphorylation

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Abstract
Stimulation with the chemokine KC induces an autocrine response in mouse astrocytes. A requirement for NF‐κB was established for KC self‐induction. NF‐κB inhibitors, p65 antisense oligonucleotides, or dominant‐negative IκBα inhibited this autocrine response. Mutation of a specific κB site in the KC promoter also blocked KC self‐induction. Chromatin immunoprecipitation and in vivo footprinting confirmed the direct binding of NF‐κB to the KC promoter. However, neither NF‐κB nuclear translocation, increased IκB degradation, nor upregulation of NF‐κB DNA binding activity was observed after KC stimulation. Reporter gene assays demonstrated KC‐upregulated NF‐κB transcriptional activity, and this effect was inhibited by dominant‐negative IκBα. Accumulation of NF‐κB was noted within the nucleus in the presence of nuclear export inhibitor leptomycin B, demonstrating constitutive shuttling of NF‐κB between the cytoplasm and nucleus. Blocking NF‐κB shuttling inhibited KC transcription. KC induced p65 phosphorylation, which was critical for NF‐κB activation as determined with the Gal‐4‐p65 fusion protein and mutation of p65 phosphorylation sites. In conclusion, low‐level nuclear NF‐κB is essential for KC self‐induction, and this effect is mediated by shuttling and phosphorylation of NF‐κB. The results outline a novel mechanism for NF‐κB participation in transcription regulation.