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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.


Related in: MedlinePlus

Effect of PKC inhibitors on LPS induced NO production in RAW 264.7 cells. Cells were treated with vehicle (control) or LPS (1 μg/ml) in the presence or absence of PKC inhibitors Go 6978 (Go, 10 μM) or bisindolylemaleimide (Bis, 20 μM) for 24 hours. The culture medium was then harvested and assayed for nitrite content by Greiss assay. The data show that only Bis was able to inhibit LPS-stimulated RAW cell NO production. Results are expressed mean ± SEM, ***p < 0.001, n = 9.
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Figure 4: Effect of PKC inhibitors on LPS induced NO production in RAW 264.7 cells. Cells were treated with vehicle (control) or LPS (1 μg/ml) in the presence or absence of PKC inhibitors Go 6978 (Go, 10 μM) or bisindolylemaleimide (Bis, 20 μM) for 24 hours. The culture medium was then harvested and assayed for nitrite content by Greiss assay. The data show that only Bis was able to inhibit LPS-stimulated RAW cell NO production. Results are expressed mean ± SEM, ***p < 0.001, n = 9.

Mentions: Effects on PKC isoforms degradation have been reported following PMA treatment at the concentration of PMA used in this study (50 ng/ml) [28]. The induction of NOS2 activity upon activation of PKC by LPS stimulation has also been demonstrated previously using a non-selective inhibitor of all PKC isoforms [16]. However, the role of specific PKC isoforms has remained unclear. The MALY peptides have been reported to mimic the PKC variable regions 1 and 2 (V1-2). These regions are necessary for binding PKC to the receptors for activated C kinase (RACK) and thereby prevent nuclear-cytoplasmic translocation of specific PKC isoforms [29]. The addition of a TAT sequence (GGGGYGRKKRRQRRR-GGGG) to the MALY peptides ensures that they are transported into the nucleus, an important facet for some PKC enzymes. Pre-treatment with these translocation inhibitor-peptides had no effect on NO production in these cells (Table 1). In addition, the PKC inhibitor Gö 6983 (Go, 10 μM) had no effect on LPS-stimulated NO production (Fig. 4).


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)

Effect of PKC inhibitors on LPS induced NO production in RAW 264.7 cells. Cells were treated with vehicle (control) or LPS (1 μg/ml) in the presence or absence of PKC inhibitors Go 6978 (Go, 10 μM) or bisindolylemaleimide (Bis, 20 μM) for 24 hours. The culture medium was then harvested and assayed for nitrite content by Greiss assay. The data show that only Bis was able to inhibit LPS-stimulated RAW cell NO production. Results are expressed mean ± SEM, ***p < 0.001, n = 9.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Effect of PKC inhibitors on LPS induced NO production in RAW 264.7 cells. Cells were treated with vehicle (control) or LPS (1 μg/ml) in the presence or absence of PKC inhibitors Go 6978 (Go, 10 μM) or bisindolylemaleimide (Bis, 20 μM) for 24 hours. The culture medium was then harvested and assayed for nitrite content by Greiss assay. The data show that only Bis was able to inhibit LPS-stimulated RAW cell NO production. Results are expressed mean ± SEM, ***p < 0.001, n = 9.
Mentions: Effects on PKC isoforms degradation have been reported following PMA treatment at the concentration of PMA used in this study (50 ng/ml) [28]. The induction of NOS2 activity upon activation of PKC by LPS stimulation has also been demonstrated previously using a non-selective inhibitor of all PKC isoforms [16]. However, the role of specific PKC isoforms has remained unclear. The MALY peptides have been reported to mimic the PKC variable regions 1 and 2 (V1-2). These regions are necessary for binding PKC to the receptors for activated C kinase (RACK) and thereby prevent nuclear-cytoplasmic translocation of specific PKC isoforms [29]. The addition of a TAT sequence (GGGGYGRKKRRQRRR-GGGG) to the MALY peptides ensures that they are transported into the nucleus, an important facet for some PKC enzymes. Pre-treatment with these translocation inhibitor-peptides had no effect on NO production in these cells (Table 1). In addition, the PKC inhibitor Gö 6983 (Go, 10 μM) had no effect on LPS-stimulated NO production (Fig. 4).

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