Functional link between TNF biosynthesis and CaM-dependent activation of inducible nitric oxide synthase in RAW 264.7 macrophages

Journal Article · Wed Jan 18 04:00:00 EST 2006 · American Journal of Physiology. Cell Physiology, 290(6):C1512-C1520

DOI:https://doi.org/10.1152/ajpcell.00527.2005· OSTI ID:877553

Weber, Thomas J; Smallwood, Heather S; Kathmann, Loel E; Markillie, Lye MENG; Squier, Thomas C; Thrall, Brian D

Inflammatory responses stimulated by bacterial endotoxin (lipopolysaccharide, LPS) involve calcium-mediated signaling, yet the cellular sensors that determine cell fate in response to LPS remain poorly understood. We report that exposure of RAW 264.7 macrophage-like cells to LPS induces a rapid increase in calmodulin (CaM) abundance, which is associated with the modulation of the inflammatory response. Increases in CaM abundance precedes nuclear localization of key transcription factors (i.e., NFκB p65 subunit, phospho-c-Jun, and Sp1) and subsequent increases in the pro-inflammatory cytokine tumor necrosis factor α (TNF) and inducible nitric oxide synthase (iNOS). Cellular apoptosis following LPS challenge is blocked following inhibition of iNOS activity, whether accomplished using the pharmacological inhibitor 1400W, through gene silencing of TNFα, or by increasing the level of cellular CaM by stable transfection. Increasing CaM expression also results in reductions in the cellular release of TNFα and iNOS, and activation of their transcriptional regulators, indicating the level of available CaM plays a key role in determining the expression of the pro-inflammatory and pro-apoptotic cascade during cellular activation by LPS. These results indicate a previously unrecognized central role for CaM in maintaining cellular homeostasis in response to LPS, such that under resting conditions cellular concentrations of CaM are sufficient to inhibit the biosynthesis of proinflammatory mediators associated with macrophage activation. Although CaM and iNOS protein levels are coordinately increased as part of the oxidative burst, limiting cellular concentrations of CaM due to association with iNOS (and other high-affinity binders) commit the cell to an unchecked inflammatory cascade leading to apoptosis.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 877553
• Report Number(s): PNNL-SA-47434
• Journal Information: American Journal of Physiology. Cell Physiology, 290(6):C1512-C1520, Journal Name: American Journal of Physiology. Cell Physiology, 290(6):C1512-C1520
• Country of Publication: United States
• Language: English