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GROWTH, DIFFERENTIATION, AND APOPTOSIS

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

Cell Biology and Biochemistry Group, Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington

Submitted 20 October 2005 ; accepted in final form 11 January 2006

Inflammatory responses stimulated by bacterial endotoxin LPS involve Ca²⁺-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 CaM abundance, which is associated with the modulation of the inflammatory response. Increases in CaM abundance precede nuclear localization of key transcription factors (i.e., NF-B p65 subunit, phospho-c-Jun, Sp1) and subsequent increases in the proinflammatory cytokine TNF- and inducible nitric oxide synthase (iNOS). Cellular apoptosis after LPS challenge is blocked upon inhibition of iNOS activity using the pharmacological inhibitor 1400W. LPS-mediated iNOS expression and apoptosis also were inhibited by siRNA-mediated silencing of TNF induction, indicating TNF induction both precedes and is necessary for subsequent regulation of iNOS expression. Increasing the level of cellular CaM by stable transfection results in reductions in LPS-induced expression of TNF and iNOS, along with reduced activation of their transcriptional regulators and concomitant protection against apoptosis. Thus the level of CaM available for Ca²⁺-dependent signaling regulation plays a key role in determining the expression of the proinflammatory and proapoptotic 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.

inflammation; apoptosis; macrophage activation

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