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Modulation of LPS stimulated NF-kappaB mediated Nitric Oxide production by PKCepsilon and JAK2 in RAW macrophages.

Jones E, Adcock IM, Ahmed BY, Punchard NA - J Inflamm (Lond) (2007)

Bottom Line: These effects were associated with changes in p65 nuclear translocation.Furthermore, a partial inhibitory effect on LPS-induced NO release was seen with the JAK2 inhibitor AG-490 and the p38 MAPK inhibitor SB 203850.The results further define the role of NF-kappaB in LPS stimulated NO production in RAW macrophages.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Health & Biosciences, University of East London, London, UK. n.punchard@uel.ac.uk.

ABSTRACT

Background: Nuclear factor kappa B (NF-kappaB) has been shown to play an important role in regulating the expression of many genes involved in cell survival, immunity and in the inflammatory processes. NF-kappaB activation upregulates inducible nitric oxide synthase leading to enhanced nitric oxide production during an inflammatory response. NF-kappaB activation is regulated by distinct kinase pathways independent of inhibitor of kappaB kinase (IKK). Here, we examine the role of protein kinase C isoforms and janus activated kinase 2 (JAK2) activation in NF-kappaB activation and LPS-stimulated NO production.

Methods: Murine RAW 264.7 macrophages were treated with lipopolysaccharide (LPS), Phorbol 12-myristate 13-acetate (PMA) and a combination of LPS and PMA in the presence or absence of various inhibitors of PKC isoforms and JAK2. Nuclear translocation of the NF-kappaB p65 subunit, was assessed by Western blot analysis whilst NO levels were assessed by Greiss assay.

Results: LPS-stimulated NO production was attenuated by PMA whilst PMA alone did not affect NO release. These effects were associated with changes in p65 nuclear translocation. The PKCalpha, beta, gamma, delta and zeta inhibitor Gö 6983 (Go) had no effect on LPS-induced NO release. In contrast, Bisindolymalemide I (Bis), a PKC alpha, betaI, betaII, gamma, delta and epsilon isoform inhibitors completely inhibited LPS-stimulated NO production without affecting p65 nuclear translocation. Furthermore, a partial inhibitory effect on LPS-induced NO release was seen with the JAK2 inhibitor AG-490 and the p38 MAPK inhibitor SB 203850.

Conclusion: The results further define the role of NF-kappaB in LPS stimulated NO production in RAW macrophages. The data support a function for PKCepsilon, JAK2 and p38 MAPK in NF-kappaB activation following p65 nuclear import.

No MeSH data available.


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Diagram showing a pictorial representation of the conclusions from all experiments (? = areas of uncertainty).
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Figure 8: Diagram showing a pictorial representation of the conclusions from all experiments (? = areas of uncertainty).

Mentions: In conclusion, the findings of the present study demonstrate the role of NF-κB in LPS stimulated NO production in RAW cells and indicate the importance of cross-talk with other kinase pathways, namely PKCε. Furthermore, the present findings further define the involvement of PKCε and JAK2 in inducing NO production, probably through their effects on NF-κB induced NOS2 expression. Figure 8 provides a pictorial summary of these findings. However, these conclusions have been drawn on the basis of use of well characterised inhibitors, rather then actual measurement of the activity of the target proteins which would provide further confirmation of this regulatory network. The work presented here further illustrates the complex network of signalling pathways involved in modulation of NF-κB-mediated gene transcription.


Modulation of LPS stimulated NF-kappaB mediated Nitric Oxide production by PKCepsilon and JAK2 in RAW macrophages.

Jones E, Adcock IM, Ahmed BY, Punchard NA - J Inflamm (Lond) (2007)

Diagram showing a pictorial representation of the conclusions from all experiments (? = areas of uncertainty).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211292&req=5

Figure 8: Diagram showing a pictorial representation of the conclusions from all experiments (? = areas of uncertainty).
Mentions: In conclusion, the findings of the present study demonstrate the role of NF-κB in LPS stimulated NO production in RAW cells and indicate the importance of cross-talk with other kinase pathways, namely PKCε. Furthermore, the present findings further define the involvement of PKCε and JAK2 in inducing NO production, probably through their effects on NF-κB induced NOS2 expression. Figure 8 provides a pictorial summary of these findings. However, these conclusions have been drawn on the basis of use of well characterised inhibitors, rather then actual measurement of the activity of the target proteins which would provide further confirmation of this regulatory network. The work presented here further illustrates the complex network of signalling pathways involved in modulation of NF-κB-mediated gene transcription.

Bottom Line: These effects were associated with changes in p65 nuclear translocation.Furthermore, a partial inhibitory effect on LPS-induced NO release was seen with the JAK2 inhibitor AG-490 and the p38 MAPK inhibitor SB 203850.The results further define the role of NF-kappaB in LPS stimulated NO production in RAW macrophages.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Health & Biosciences, University of East London, London, UK. n.punchard@uel.ac.uk.

ABSTRACT

Background: Nuclear factor kappa B (NF-kappaB) has been shown to play an important role in regulating the expression of many genes involved in cell survival, immunity and in the inflammatory processes. NF-kappaB activation upregulates inducible nitric oxide synthase leading to enhanced nitric oxide production during an inflammatory response. NF-kappaB activation is regulated by distinct kinase pathways independent of inhibitor of kappaB kinase (IKK). Here, we examine the role of protein kinase C isoforms and janus activated kinase 2 (JAK2) activation in NF-kappaB activation and LPS-stimulated NO production.

Methods: Murine RAW 264.7 macrophages were treated with lipopolysaccharide (LPS), Phorbol 12-myristate 13-acetate (PMA) and a combination of LPS and PMA in the presence or absence of various inhibitors of PKC isoforms and JAK2. Nuclear translocation of the NF-kappaB p65 subunit, was assessed by Western blot analysis whilst NO levels were assessed by Greiss assay.

Results: LPS-stimulated NO production was attenuated by PMA whilst PMA alone did not affect NO release. These effects were associated with changes in p65 nuclear translocation. The PKCalpha, beta, gamma, delta and zeta inhibitor Gö 6983 (Go) had no effect on LPS-induced NO release. In contrast, Bisindolymalemide I (Bis), a PKC alpha, betaI, betaII, gamma, delta and epsilon isoform inhibitors completely inhibited LPS-stimulated NO production without affecting p65 nuclear translocation. Furthermore, a partial inhibitory effect on LPS-induced NO release was seen with the JAK2 inhibitor AG-490 and the p38 MAPK inhibitor SB 203850.

Conclusion: The results further define the role of NF-kappaB in LPS stimulated NO production in RAW macrophages. The data support a function for PKCepsilon, JAK2 and p38 MAPK in NF-kappaB activation following p65 nuclear import.

No MeSH data available.


Related in: MedlinePlus