Low nuclear levels of nuclear factor-kappa B are essential for KC self-induction in astrocytes: requirements for shuttling and phosphorylation.
Stimulation with the chemokine KC induces an autocrine response in mouse astrocytes. A requirement for NF-kappa B was established for KC self-induction. NF-kappa B inhibitors, p65 antisense oligonucleotides, or dominant-negative Ikappa Balpha inhibited this autocrine response. Mutation of a specific kappa B site in the KC promoter also blocked KC self-induction. Chromatin immunoprecipitation and in vivo footprinting confirmed the direct binding of NF-kappa B to the KC promoter. However, neither NF-kappa B nuclear translocation, increased Ikappa B degradation, nor upregulation of NF-kappa B DNA binding activity was observed after KC stimulation. Reporter gene assays demonstrated KC-upregulated NF-kappa B transcriptional activity, and this effect was inhibited by dominant-negative IkappaBalpha. Accumulation of NF-kappaB was noted within the nucleus in the presence of nuclear export inhibitor leptomycin B, demonstrating constitutive shuttling of NF-kappa B between the cytoplasm and nucleus. Blocking NF-kappa B shuttling inhibited KC transcription. KC induced p65 phosphorylation, which was critical for NF-kappa B activation as determined with the Gal-4-p65 fusion protein and mutation of p65 phosphorylation sites. In conclusion, low-level nuclear NF-kappa B is essential for KC self-induction, and this effect is mediated by shuttling and phosphorylation of NF-kappa B. The results outline a novel mechanism for NF-kappa B participation in transcription regulation.
Zhai
Qiwei
Q
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Luo
Yi
Y
Zhang
Ye
Y
Berman
Michael A
MA
Dorf
Martin E
ME
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D054360:Chemokine CXCL1; D018925:Chemokines; D019743:Chemokines, CXC; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000096:biosynthesis* / Q000235:genetics; D010766:Phosphorylation; D014158:Transcription, Genetic / Q000502:physiology
D054360:Chemokine CXCL1; D018925:Chemokines; D019743:Chemokines, CXC; C475895:Cxcl1 protein, mouse; D016207:Cytokines; D016328:NF-kappa B; C074302:keratinocyte-derived chemokines