Abstract
In macrophages, nuclear factor kappa B (NF-κB) has been shown to transactivate the promoters of many cytokines, including tumor necrosis factor-α (TNF-α). We have used the —510 κB binding site from the murine TNF-α promoter to assay the induction of NF-κB in murine macrophages by various stimuli. A basal level of NF-κB activity in murine macrophages was detectable, and this activity was enhanced by treatment of these cells with lipopolysaccharide (LPS) or interleukin-2 (IL-2). Interferon-γ (IF N-γ), an important regulator of macrophage gene expression, significantly enhanced NF-κB activity and altered the apparent molecular weight of the NF-κB1-like proteins in LPS-stimulated and IL-2-stimulated murine macrophages. The NRD (NF-κB/Rel/Dorsal) complexes induced by LPS and IFN-γ were further characterized by addition of antisera to electrophoretic mobility shift assay (EMSA) reaction mixtures. NF-κB1/p50 was a component of all complexes, whereas RelA/p65 was present in the IFN-γ/LPS-stimulated activity. IFN-γ priming or treatment with LPS for 19 h resulted in an upregulation of the larger species of NF-κB1/p50. In addition, regulation of the two pools of NF-κB1/p50 by IFN-γ was confirmed by Western immunoblot analysis of cytosolic and nuclear extracts. This is the first demonstration of the presence of two pools of NF-κB1/p50 differentially regulated in response to cytokine activation of macrophages.
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