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Specific PKC isoforms regulate LPS-stimulated iNOS induction in murine microglial cells
Jie Wen1, Rachel Ribeiro, Yumin Zhang
1Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA.
Background:
Excessive production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) in reactive microglia is a major contributor to initiation/exacerbation of inflammatory and degenerative neurological diseases. Previous studies have indicated that activation of protein kinase C (PKC) can lead to iNOS induction. Because of the existence of various PKC isoforms and the ambiguous specificity of PKC inhibitors, it is unclear whether all PKC isoforms or a specific subset are involved in the expression of iNOS by reactive microglia. In this study, we employed molecular approaches to characterize the role of each specific PKC isoform in the regulation of iNOS expression in murine microglia.
Methods:
Induction of iNOS in response to bacterial endotoxin lipopolysaccharide (LPS) was measured in BV-2 murine microglia treated with class-specific PKC inhibitors, or transfected with siRNA to silence specific PKC isoforms. iNOS expression and MAPK phosphorylation were evaluated by western blot. The role of NF-κB in activated microglia was examined by determining NF-κB transcriptional response element- (TRE-) driven, promoter-mediated luciferase activity.
Results:
Murine microglia expressed high levels of nPKCs, and expressed relatively low levels of cPKCs and aPKCs. All PKC inhibitors attenuated induction of iNOS in LPS-activated microglia. Knockdown of PKC δ and PKC β attenuated ERK1/2 and p38 phosphorylation, respectively, and blocked NF-κB activation that leads to the expression of iNOS in reactive microglia.
Conclusions:
Our results identify PKC δ and β as the major PKC isoforms regulating iNOS expression in reactive microglia. The signaling pathways mediated by PKC involve phosphorylation of distinct MAPKs and activation of NF-κB. These results may help in the design of novel and selective PKC inhibitors for the treatment of many inflammatory and neurological diseases in which production of NO plays a pathogenic role.
Insights
Protein Kinase C delta (PKCδ) and beta (PKCβ) isoforms are key regulators of inducible nitric oxide synthase (iNOS) expression in reactive microglia. Targeting these specific PKC isoforms may offer novel therapeutic strategies for inflammatory and neurological diseases.
Area of Science:
- Neuroscience
- Immunology
- Molecular Biology
Background:
- Reactive microglia contribute to neurological diseases via excessive nitric oxide (NO) production by inducible nitric oxide synthase (iNOS).
- Protein Kinase C (PKC) activation is linked to iNOS induction, but specific isoform roles remain unclear.
- Understanding specific PKC isoform involvement is crucial for developing targeted therapies.
Purpose of the Study:
- To elucidate the role of individual PKC isoforms in regulating iNOS expression in murine microglia.
- To identify the specific signaling pathways downstream of PKC involved in iNOS induction.
Main Methods:
- Utilized BV-2 murine microglia treated with specific PKC inhibitors and siRNA for isoform knockdown.
- Assessed iNOS expression and MAPK phosphorylation via Western blot.
- Investigated NF-κB activation using luciferase reporter assays.
Main Results:
- All tested PKC inhibitors reduced iNOS induction in lipopolysaccharide (LPS)-activated microglia.
- Knockdown of PKCδ and PKCβ significantly attenuated ERK1/2 and p38 phosphorylation, respectively.
- PKCδ and PKCβ inhibition blocked NF-κB activation, thereby suppressing iNOS expression.
Conclusions:
- PKCδ and PKCβ are identified as the primary PKC isoforms regulating iNOS expression in reactive microglia.
- PKC-mediated signaling involves distinct MAPK phosphorylation and NF-κB activation.
- These findings support the development of selective PKC inhibitors for treating NO-related inflammatory and neurological disorders.
