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Phorbol ester synergistically increases interferon-gamma-induced nitric oxide synthesis in murine microglial cells
1Department of Microbiology and Immunology, School of Medicine, Wonkwang University, Iri, Korea.
Abstract:
The effect of phorbol ester on the synthesis of nitric oxide (NO) in murine microglial cells was examined. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, alone had no effect, whereas PMA with recombinant interferon (rIFN)-gamma synergistically increased NO synthesis in murine microglial cells. The maximal effect of PMA on NO synthesis increase always fitted with the range for full activation of PKC in these cells. The increase of NO synthesis was reflected as increased amount of immunologic NO synthase (iNOS) mRNA detected by Northern blotting. Treatment with PKC inhibitors such as staurosporine or polymyxin B decreased rIFN-gamma-plus-PMA-stimulated NO synthesis. Further, prolonged incubation of the cells with PMA, which down-regulates PKC activity, abolished the synergistic cooperative effect with IFN-gamma. NG-monomethyl-L-arginine monohydrate, an analogue of L-arginine, and arginase inhibited rIFN-gamma-plus-PMA-induced NO production in murine microglial cells. On the basis of these observations, we conclude that PKC might not be involved in the expression of iNOS, but instead, might be involved in the posttranscriptional modification of iNOS mRNA.
Insights
Phorbol ester (PMA) combined with interferon-gamma synergistically boosts nitric oxide (NO) synthesis in microglial cells. Protein kinase C (PKC) activation appears crucial for this NO production, potentially through post-transcriptional regulation of iNOS mRNA.
Area of Science:
- Immunology
- Neuroscience
- Cell Biology
Background:
- Microglial cells play a critical role in neuroinflammation.
- Nitric oxide (NO) is a key mediator in immune responses.
- Protein kinase C (PKC) is implicated in cellular signaling pathways.
Purpose of the Study:
- To investigate the role of phorbol ester (PMA), a PKC activator, in regulating nitric oxide (NO) synthesis in murine microglial cells.
- To elucidate the signaling mechanisms underlying NO production, particularly the involvement of PKC and interferon-gamma (IFN-γ).
Main Methods:
- Murine microglial cells were treated with PMA and/or rIFN-γ.
- Nitric oxide synthesis was measured.
- Immunologic NO synthase (iNOS) mRNA levels were assessed using Northern blotting.
- PKC activity was modulated using inhibitors (staurosporine, polymyxin B) and prolonged PMA incubation.
- L-arginine analogues and arginase were used to assess NO production pathways.
Main Results:
- PMA alone did not affect NO synthesis, but synergistically increased it with rIFN-γ.
- Maximal PMA effect correlated with full PKC activation.
- Increased NO synthesis corresponded to higher iNOS mRNA levels.
- PKC inhibitors and prolonged PMA treatment (down-regulating PKC) reduced NO synthesis.
- Inhibition of L-arginine pathway components decreased NO production.
Conclusions:
- PKC activation is essential for the synergistic increase in NO synthesis induced by PMA and rIFN-γ in microglial cells.
- PKC's role may lie in the post-transcriptional modification of iNOS mRNA rather than its direct expression.
- These findings highlight a complex regulatory mechanism of NO production in microglia involving PKC signaling.