Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NF-kappaB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells

Biochem Biophys Res Commun. 2004 Feb 13;314(3):695-703. doi: 10.1016/j.bbrc.2003.12.153.

Abstract

Ca(2+) and Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN) have been known to play crucial roles in immune response and inflammation. Using mouse peritoneal macrophages and RAW 264.7 macrophage cells, we demonstrated that LPS mobilized intracellular free Ca(2+) and induced CN phosphatase activity. iNOS expression and NO secretion in response to LPS were suppressed by Ca(2+) antagonists (TMB-8, BAPTA/AM, and nifedipine) and CN inhibitor (cyclosporin A). Transient expression of constitutively active CN in mouse peritoneal macrophages and RAW 264.7 macrophages strongly activated NF-kappaB, a key mediator of iNOS expression. We also found that CN mediates NF-kappaB activation via IkappaB-alpha hyperphosphorylation and degradation. Overexpression of dominant negative mutant of IKKalpha and -beta demonstrates that only IKKbeta is the target for CN. These results indicate that CN is required for full iNOS expression and the effective activation of NF-kappaB in RAW 264.7 and peritoneal macrophages.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcineurin / metabolism*
  • Calcineurin Inhibitors
  • Calcium / metabolism
  • Cell Line
  • Chelating Agents / pharmacology
  • Cyclosporine / pharmacology
  • Enzyme Activation / drug effects
  • Gene Expression Regulation, Enzymologic / drug effects
  • Genes, Reporter / drug effects
  • Genes, Reporter / genetics
  • I-kappa B Kinase
  • Lipopolysaccharides / pharmacology*
  • Macrophages, Peritoneal / drug effects
  • Macrophages, Peritoneal / enzymology*
  • Mice
  • Mice, Inbred C57BL
  • NF-kappa B / antagonists & inhibitors
  • NF-kappa B / genetics
  • NF-kappa B / metabolism*
  • Nitric Oxide / biosynthesis
  • Nitric Oxide Synthase / biosynthesis*
  • Nitric Oxide Synthase / genetics
  • Nitric Oxide Synthase Type II
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Transfection

Substances

  • Calcineurin Inhibitors
  • Chelating Agents
  • Lipopolysaccharides
  • NF-kappa B
  • Nitric Oxide
  • Cyclosporine
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Nos2 protein, mouse
  • Protein-Serine-Threonine Kinases
  • Chuk protein, mouse
  • I-kappa B Kinase
  • Ikbkb protein, mouse
  • Ikbke protein, mouse
  • Calcineurin
  • Calcium