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

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 phosph...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2004-02, Vol.314 (3), p.695-703
Main Authors: Kim, YoungHee, Moon, Ji Sun, Lee, Kyoung Soon, Park, Sun Young, Cheong, JaeHun, Kang, Ho Sung, Lee, Hak Young, Kim, Han Do
Format: Article
Language:English
Subjects:
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
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Summary: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.
ISSN:0006-291X