Murine inhibitory protein-kappa B alpha negatively regulates kappa B-dependent transcription in lipopolysaccharide-stimulated RAW 264.7 macrophages.

The potential role of the inhibitory protein (I)-kappa B alpha gene in control of LPS-dependent transcription has been investigated in the murine macrophage cell line RAW 264.7. LPS-induced transcription in macrophages is believed to involve activation of members of the Rel homology family of transcription factors, and may be negatively regulated by cytoplasmic inhibitor proteins collectively termed I-kappa Bs. To evaluate the role of I-kappa Bs in LPS-stimulated macrophages, murine I-kappa B alpha (ml-kappa B alpha) has been expressed as a glutathione-S-transferase (GST) fusion protein and examined for its ability to control kappa B binding activities in nuclear extracts from LPS-treated RAW 264.7 macrophages. ml-kappa B alpha-GST inhibited LPS-induced kappa B binding activity from RAW 264.7 cells in a phosphorylation-dependent fashion, but did not affect IFN-alpha-induced IFN stimulus response element binding. Recombinant I-kappa B alpha inhibited kappa B motif binding by nuclear factor-kappa B1, RelA, and c-Rel as indicated by studies using UV radiation-induced covalent cross-linking to a bromodeoxyuridine-substituted kappa B oligonucleotide. Transfection of macrophages with an expression vector encoding ml-kappa B alpha inhibited LPS-stimulated transcription driven by a 243-bp promoter sequence obtained from the 5' flanking region of the murine IP-10 gene. This promoter sequence contains two kappa B motifs that have been shown to be critical to LPS-dependent reporter gene transcription. The kappa B sites seem to be the specific target of I-kappa B alpha function, as reporter gene transcription driven by these motifs in the context of a heterologous thymidine-kinase promoter (TK) also was inhibited by co-transfection with ml-kappa B alpha. These observations indicate that ml-kappa B alpha is capable of controlling kappa B-dependent transcription in LPS-stimulated murine macrophages.