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Potential role of PKR in double-stranded RNA-induced macrophage activation
L B Maggi1, M R Heitmeier, D Scheuner
1The Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 South Grand Blvd, St Louis, MO 63104, USA.
Abstract:
In this study, the role of the double-stranded (ds) RNA-dependent protein kinase (PKR) in macrophage activation was examined. dsRNA [polyinosinic:polycytidylic acid (poly IC)]-stimulated inducible nitric oxide synthase, interleukin (IL)-1alpha and IL-1beta mRNA expression, nitrite formation and IL-1 release are attenuated in RAW264.7 cells stably expressing dominant negative (dn) mutants of PKR. The transcriptional regulator nuclear factor (NF)-kappaB is activated by dsRNA, and appears to be required for dsRNA-induced macrophage activation. While dnPKR mutants prevent macrophage activation, they fail to attenuate dsRNA-induced IkappaB degradation or NF-kappaB nuclear localization. The inhibitory actions of dnPKR on dsRNA-induced macrophage activation can be overcome by treatment with interferon (IFN)-gamma, an event associated with PKR degradation. Furthermore, dsRNA + IFN-gamma stimulate inducible nitric oxide synthase expression, IkappaB degradation and NF-kappaB nuclear localization to similar levels in macrophages isolated from PKR(-/-) and PKR(+/+) mice. These findings indicate that both NF-kappaB and PKR are required for dsRNA-induced macrophage activation; however, dsRNA-induced NF-kappaB activation occurs by PKR-independent mechanisms in macrophages. In addition, the PKR dependence of dsRNA-induced macrophage activation can be overcome by IFN-gamma.
Insights
Double-stranded RNA-dependent protein kinase (PKR) is crucial for macrophage activation, but its role in nuclear factor-kappaB (NF-kappaB) activation is independent. Interferon-gamma can overcome PKR
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Double-stranded RNA (dsRNA) activates macrophages through various pathways.
- Protein kinase R (PKR) is a key mediator of cellular responses to dsRNA.
- Nuclear factor-kappaB (NF-kappaB) is a critical transcription factor involved in immune responses.
Purpose of the Study:
- To investigate the role of PKR in dsRNA-induced macrophage activation.
- To elucidate the relationship between PKR, NF-kappaB, and macrophage activation.
- To determine the effect of interferon-gamma (IFN-gamma) on dsRNA-induced macrophage activation in the context of PKR.
Main Methods:
- Utilized RAW264.7 macrophage cell lines stably expressing dominant-negative PKR mutants.
- Assessed mRNA expression of inducible nitric oxide synthase (iNOS), IL-1alpha, and IL-1beta.
- Measured nitrite formation, IL-1 release, IkappaB degradation, and NF-kappaB nuclear localization.
- Compared responses in macrophages from PKR knockout (PKR-/-) and wild-type (PKR+/+) mice.
Main Results:
- Dominant-negative PKR mutants attenuated dsRNA-induced iNOS, IL-1alpha, IL-1beta expression, nitrite formation, and IL-1 release.
- dsRNA-induced IkappaB degradation and NF-kappaB nuclear localization were not prevented by dominant-negative PKR mutants.
- Interferon-gamma treatment overcame the inhibitory effects of dominant-negative PKR and led to similar iNOS expression and NF-kappaB activation in PKR-/- and PKR+/+ macrophages.
- These results suggest dsRNA-induced NF-kappaB activation is PKR-independent.
Conclusions:
- Both NF-kappaB and PKR are necessary for dsRNA-induced macrophage activation.
- dsRNA-induced NF-kappaB activation proceeds through a PKR-independent pathway.
- Interferon-gamma can override the PKR-dependent requirement for dsRNA-induced macrophage activation.