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

Abstract

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 aremore » 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.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
877553
Report Number(s):
PNNL-SA-47434
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: American Journal of Physiology. Cell Physiology, 290(6):C1512-C1520
Country of Publication:
United States
Language:
English

Citation Formats

Weber, Thomas J, Smallwood, Heather S, Kathmann, Loel E, Markillie, Lye MENG, Squier, Thomas C, and Thrall, Brian D. Functional link between TNF biosynthesis and CaM-dependent activation of inducible nitric oxide synthase in RAW 264.7 macrophages. United States: N. p., 2006. Web. doi:10.1152/ajpcell.00527.2005.
Weber, Thomas J, Smallwood, Heather S, Kathmann, Loel E, Markillie, Lye MENG, Squier, Thomas C, & Thrall, Brian D. Functional link between TNF biosynthesis and CaM-dependent activation of inducible nitric oxide synthase in RAW 264.7 macrophages. United States. doi:10.1152/ajpcell.00527.2005.
Weber, Thomas J, Smallwood, Heather S, Kathmann, Loel E, Markillie, Lye MENG, Squier, Thomas C, and Thrall, Brian D. Wed . "Functional link between TNF biosynthesis and CaM-dependent activation of inducible nitric oxide synthase in RAW 264.7 macrophages". United States. doi:10.1152/ajpcell.00527.2005.
@article{osti_877553,
title = {Functional link between TNF biosynthesis and CaM-dependent activation of inducible nitric oxide synthase in RAW 264.7 macrophages},
author = {Weber, Thomas J and Smallwood, Heather S and Kathmann, Loel E and Markillie, Lye MENG and Squier, Thomas C and Thrall, Brian D},
abstractNote = {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.},
doi = {10.1152/ajpcell.00527.2005},
journal = {American Journal of Physiology. Cell Physiology, 290(6):C1512-C1520},
number = ,
volume = ,
place = {United States},
year = {Wed Jan 18 00:00:00 EST 2006},
month = {Wed Jan 18 00:00:00 EST 2006}
}
  • Carabrol, isolated from Carpesium macrocephalum, showed anti-inflammatory potential in LPS-induced RAW 264.7 murine macrophages. In present study, carabrol demonstrated the inhibitory activity on pro-inflammatory cytokines such as IL-1{beta}, IL-6 and TNF-{alpha}. In addition, mRNA and protein levels of iNOS and COX-2 were reduced by carabrol. Molecular analysis revealed that these suppressive effects were correlated with the inactivation of p38 and JNK via inhibition of NF-{kappa}B activation. Immunoblotting showed that carabrol suppressed LPS-induced degradation of I-{kappa}B{alpha} and decreased nuclear translocation of p65. Taken together, these results suggest that carabrol can be a modulator of pro-inflammatory signal transduction pathway in RAW 264.7more » cells.« less
  • Crotafuran B, a natural pterocarpanoid isolated from Crotalaria pallida, inhibited the lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (IC{sub 5} 16.4 {+-} 0.7 {mu}M) and inducible nitric oxide synthase (iNOS) protein and mRNA expression (IC{sub 5} 11.5 {+-} 0.6 {mu}M and 11.8 {+-} 2.2 {mu}M, respectively), but not via its cytotoxicity or the inhibition of iNOS enzyme activity, in RAW 264.7 macrophages. Crotafuran B also reduced the iNOS promoter activity (IC{sub 5} 13.4 {+-} 0.1 {mu}M) in piNOS-LUC-transfected cells. Crotafuran B treatment inhibited the p65 nuclear translocation and the nuclear factor-{kappa}B (NF-{kappa}B) DNA binding activity in LPS-activated macrophages. Crotafuran B alsomore » reduced the NF-{kappa}B transcriptional activity in pNF-{kappa}B-LUC-transfected cells. Crotafuran B had no effect on the LPS-induced phosphorylation of inhibitory {kappa}B{alpha} (I{kappa}B{alpha}), but enhanced the cellular level of I{kappa}B{alpha} that rebounded to the basal levels and increased the I{kappa}B{alpha} mRNA expression. These results indicate that the crotafuran B inhibition of NO production involves a decrease in the iNOS gene expression via the inhibition of NF-{kappa}B activation through the increase in I{kappa}B{alpha} synthesis.« less
  • Chebulagic acid (CA), a natural anti-oxidant, showed potent anti-inflammatory effects in LPS-stimulated RAW 264.7, a mouse macrophage cell line. These effects were exerted via inhibition of NO and PGE{sub 2} production and down-regulation of iNOS, COX-2, 5-LOX, TNF-{alpha} and IL-6. CA inhibited NF-{kappa}B activation by LPS, and this was associated with the abrogation of I{kappa}B-{alpha} phosphorylation and subsequent decreases in nuclear p50 and p65 protein levels. Further, the phosphorylation of p38, ERK 1/2 and JNK in LPS-stimulated RAW 264.7 cells was suppressed by CA in a concentration-dependent manner. LPS-induced generation of reactive oxygen species (ROS) was also effectively inhibited bymore » CA. These results suggest that CA exerts anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophages by inhibition of NF-{kappa}B activation and MAP kinase phosphorylation.« less
  • Purpose: To explore whether antitumor immunoadjuvant OM-174 can stimulate immune cells to produce interferon-{gamma} (IFN-{gamma}) and thereby radiosensitize tumor cells. Methods and Materials: Splenocytes from BALB/c mice were stimulated by OM-174 at plasma-achievable concentrations (0.03-3 {mu}g/mL), and afterward analyzed for the expression and secretion of IFN-{gamma} by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Stimulated splenocytes were used as a source of IFN-{gamma} to radiosensitize hypoxic EMT-6 tumor cells through the cytokine-inducible isoform of nitric oxide synthase (iNOS). Results: OM-174 activated the production of IFN-{gamma} at high levels that reached 70 ng/mL in normoxia (21% oxygen) and 27more » ng/mL in tumor-relevant hypoxia (1% oxygen). This caused up to 2.1-fold radiosensitization of EMT-6 tumor cells, which was associated with the iNOS-mediated production of the radiosensitizing molecule nitric oxide, as confirmed by accumulation of its oxidative metabolite nitrite, Western blot analysis, and reverse transcriptase-polymerase chain reaction. Both iNOS activation and radiosensitization were counteracted by neutralizing antibodies against IFN-{gamma}. The same mechanism of radiosensitization through the IFN-{gamma} secretion pathway was identified for IL-12 + IL-18, which are known to mediate IFN-{gamma} responses. Hypoxia displayed a dual effect on the immune-tumor cell interaction, by downregulating the expression of the IFN-{gamma} gene while upregulating iNOS at transcriptional level. Conclusion: Immunoadjuvant OM-174 is an efficient radiosensitizer of tumor cells through activation of the IFN-{gamma} secretion pathway in immune cells. This finding indicates a rationale for combining immunostimulatory and radiosensitizing strategies and extends the potential therapeutic applications of OM-174.« less