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 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.
Kim
YoungHee
Y
Korea NanoBioTechnology Center, College of Natural Sciences, Pusan National University, 609-735, Busan, Republic of Korea.
Moon
Ji Sun
JS
Lee
Kyoung Soon
KS
Park
Sun Young
SY
Cheong
JaeHun
J
Kang
Ho Sung
HS
Lee
Hak Young
HY
Kim
Han Do
HD
D000818:Animals; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter / Q000187:drug effects / Q000235:genetics; D051550:I-kappa B Kinase; D008070:Lipopolysaccharides / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014162:Transfection
D065095:Calcineurin Inhibitors; D002614:Chelating Agents; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009569:Nitric Oxide; D016572:Cyclosporine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D017346:Protein Serine-Threonine Kinases; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; D019703:Calcineurin; D002118:Calcium