Regulation of NF-kappa B activity in murine macrophages: effect of bacterial lipopolysaccharide and phorbol ester

J Cell Physiol. 1992 Jan;150(1):204-13. doi: 10.1002/jcp.1041500127.

Abstract

Nuclear factor kappa-B (NF-kappa B) 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-kappa B activity. In this study we have investigated the mechanism of NF-kappa B induction in murine macrophages. A chloramphenicol acetyl transferase (CAT) expression vector containing multiple copies of the TNF-alpha NF-kappa B 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-kappa B 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-kappa B 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-kappa B 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-kappa B activity in these cells. These results suggest that the induction of NF-kappa B in macrophages by LPS is independent of PKC.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
  • Animals
  • Base Sequence
  • Cell Line
  • DNA
  • Down-Regulation
  • Electrophoresis, Polyacrylamide Gel
  • Isoquinolines / pharmacology
  • Kinetics
  • Lipopolysaccharides / metabolism*
  • Macrophages / metabolism*
  • Mice
  • Molecular Sequence Data
  • Molecular Weight
  • NF-kappa B / metabolism*
  • Piperazines / pharmacology
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism*
  • Tetradecanoylphorbol Acetate / pharmacology*

Substances

  • Isoquinolines
  • Lipopolysaccharides
  • NF-kappa B
  • Piperazines
  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
  • DNA
  • Protein Kinase C
  • Tetradecanoylphorbol Acetate