Ca 2+/calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NF-κB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells

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-κB, a key mediator of iNOS expression. We also found that CN mediates NF-κB activation via IκB-α hyperphosphorylation and degradation. Overexpression of dominant negative mutant of IKKα and -β demonstrates that only IKKβ is the target for CN. These results indicate that CN is required for full iNOS expression and the effective activation of NF-κB in RAW 264.7 and peritoneal macrophages.