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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

a. The effect of PMA on LPS stimulated NO production in RAW 264.7 cells. RAW cells were stimulated for 24 hr with either vehicle alone (control), 1 μg/ml LPS, 50 ng/ml PMA or 1 μg/ml LPS with 50 ng/ml PMA. NO levels were assessed by Greiss assay. LPS alone significantly increased NO production whereas PMA alone had no effect. PMA significantly inhibited LPS -stimulated NO production. Figure 1b (inset) displays the concentration-response curve for LPS-stimulated NO production over 24 hrs. Results are expressed as mean ± SEM; +++p < 0.001 vs control, ***p < 0.001 vs LPs-stimulated; n = 6 for the effects of PMA and n = 9 for the concentration-response curve.
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Figure 1: a. The effect of PMA on LPS stimulated NO production in RAW 264.7 cells. RAW cells were stimulated for 24 hr with either vehicle alone (control), 1 μg/ml LPS, 50 ng/ml PMA or 1 μg/ml LPS with 50 ng/ml PMA. NO levels were assessed by Greiss assay. LPS alone significantly increased NO production whereas PMA alone had no effect. PMA significantly inhibited LPS -stimulated NO production. Figure 1b (inset) displays the concentration-response curve for LPS-stimulated NO production over 24 hrs. Results are expressed as mean ± SEM; +++p < 0.001 vs control, ***p < 0.001 vs LPs-stimulated; n = 6 for the effects of PMA and n = 9 for the concentration-response curve.

Mentions: Constitutive NO production by RAW 264.7 macrophages (Fig. 1) was at the lower limit of the sensitivity of the assay (3 μM,). LPS induced a concentration-dependent increase in NO production with a maximum response at 50 μg/ml at 24 h (Figure 1b.). For subsequent experiments, LPS (1 μg/ml) was chosen for its ability to stimulate high levels of NO production whilst not possessing the significant (p < 0.001) toxicity seen with 10 and 50 μg/ml (38% and 51% reduction in viability respectively).


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)

a. The effect of PMA on LPS stimulated NO production in RAW 264.7 cells. RAW cells were stimulated for 24 hr with either vehicle alone (control), 1 μg/ml LPS, 50 ng/ml PMA or 1 μg/ml LPS with 50 ng/ml PMA. NO levels were assessed by Greiss assay. LPS alone significantly increased NO production whereas PMA alone had no effect. PMA significantly inhibited LPS -stimulated NO production. Figure 1b (inset) displays the concentration-response curve for LPS-stimulated NO production over 24 hrs. Results are expressed as mean ± SEM; +++p < 0.001 vs control, ***p < 0.001 vs LPs-stimulated; n = 6 for the effects of PMA and n = 9 for the concentration-response curve.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211292&req=5

Figure 1: a. The effect of PMA on LPS stimulated NO production in RAW 264.7 cells. RAW cells were stimulated for 24 hr with either vehicle alone (control), 1 μg/ml LPS, 50 ng/ml PMA or 1 μg/ml LPS with 50 ng/ml PMA. NO levels were assessed by Greiss assay. LPS alone significantly increased NO production whereas PMA alone had no effect. PMA significantly inhibited LPS -stimulated NO production. Figure 1b (inset) displays the concentration-response curve for LPS-stimulated NO production over 24 hrs. Results are expressed as mean ± SEM; +++p < 0.001 vs control, ***p < 0.001 vs LPs-stimulated; n = 6 for the effects of PMA and n = 9 for the concentration-response curve.
Mentions: Constitutive NO production by RAW 264.7 macrophages (Fig. 1) was at the lower limit of the sensitivity of the assay (3 μM,). LPS induced a concentration-dependent increase in NO production with a maximum response at 50 μg/ml at 24 h (Figure 1b.). For subsequent experiments, LPS (1 μg/ml) was chosen for its ability to stimulate high levels of NO production whilst not possessing the significant (p < 0.001) toxicity seen with 10 and 50 μg/ml (38% and 51% reduction in viability respectively).

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