Regulation of NF‐κB activity in murine macrophages: Effect of bacterial lipopolysaccharide and phorbol ester

Nuclear factor kappa‐B (NF‐κB) has been shown to play an important role in LPS‐mediated induction of several genes in macrophages. Several studies have implicated protein kinase C (PKC) or cAMP‐dependent protein kinase in the regulation of NF‐κB activity. In this study we have investigated the mechanism of NF‐κB induction in murine macrophages. A chloramphenicol acetyl transferase (CAT) expression vector containing multiple copies of the TNF‐α NF‐κB element was transfected into the RAW264 macrophage‐like cell line and assessed for inducible CAT activity. LPS treatment of the transfected cells resulted in a significant induction of CAT activity. CAT activity was not induced by treatment with phorbol myristate acetate (PMA) or the cAMP analogue 8‐bromo cAMP. To further study NF‐κB induction, nuclear extracts were prepared from RAW264 cells. Extracts from RAW264 cells that were treated from 30 min to 2 hr with LPS had a significant increase in NF‐κB binding activity as determined by the electrophoresis mobility shift assay (EMSA). Treatment of these cells from 30 min to 2 hr with PMA did not result in such binding activity. U.V. crosslinking analysis of the DNA‐binding activity confirmed these results and indicated that LPS induced a 55 KD DNA‐binding protein. Induction of this NF‐κB binding activity was not inhibited by pretreatment with the PKC inhibitor H‐7. H‐7 did inhibit induction of TPA responsive element binding by either LPS or PMA. Prolonged exposure to phorbol ester, a treatment which down‐regulates PKC, had no effect on LPS induction of NF‐κB activity in these cells. These results suggest that the induction of NF‐κB in macrophages by LPS is independent of PKC.