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Activation of nuclear factor kappaB and Bcl-x survival gene expression by nerve growth factor requires tyrosine phosphorylation of IkappaBalpha.

Bui NT, Livolsi A, Peyron JF, Prehn JH - J. Cell Biol. (2001)

Bottom Line: Moreover, in contrast to TNF-alpha, NGF failed to phosphorylate IkappaBalpha at serine residue 32, but instead caused significant tyrosine phosphorylation.Conversely, overexpression of a dominant negative mutant of TNF receptor-associated factor-6 blocked TNF-alpha-, but not NGF-induced NFkappaB activation.We conclude that NGF and TNF-alpha induce different signaling pathways in neurons to activate NFkappaB and bcl-x gene expression.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Center for Clinical Research, Research Group "Apoptosis and Cell Death,", D-48149 Münster, Germany.

ABSTRACT
NGF has been shown to support neuron survival by activating the transcription factor nuclear factor-kappaB (NFkappaB). We investigated the effect of NGF on the expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein. Treatment of rat pheochromocytoma PC12 cells, human neuroblastoma SH-SY5Y cells, or primary rat hippocampal neurons with NGF (0.1-10 ng/ml) increased the expression of bcl-xL mRNA and protein. Reporter gene analysis revealed a significant increase in NFkappaB activity after treatment with NGF that was associated with increased nuclear translocation of the active NFkappaB p65 subunit. NGF-induced NFkappaB activity and Bcl-xL expression were inhibited in cells overexpressing the NFkappaB inhibitor, IkappaBalpha. Unlike tumor necrosis factor-alpha (TNF-alpha), however, NGF-induced NFkappaB activation occurred without significant degradation of IkappaBs determined by Western blot analysis and time-lapse imaging of neurons expressing green fluorescent protein-tagged IkappaBalpha. Moreover, in contrast to TNF-alpha, NGF failed to phosphorylate IkappaBalpha at serine residue 32, but instead caused significant tyrosine phosphorylation. Overexpression of a Y42F mutant of IkappaBalpha potently suppressed NFG-, but not TNF-alpha-induced NFkappaB activation. Conversely, overexpression of a dominant negative mutant of TNF receptor-associated factor-6 blocked TNF-alpha-, but not NGF-induced NFkappaB activation. We conclude that NGF and TNF-alpha induce different signaling pathways in neurons to activate NFkappaB and bcl-x gene expression.

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TNF-α, but not NGF induces degradation of IκBs α and β. Degradation of IκBα and IκBβ in PC12 cells treated with (a) TNF-α (10 ng/ml) or (b) NGF (1 ng/ml) for the indicated period of time. 50 μg protein extract were separated on 12% SDS-PAGE, blotted onto nitrocellulose membrane, and IκBα or β was detected using rabbit polyclonal antibodies. Membranes were stripped and probed with an α-tubulin mouse monoclonal antibody as control for equal sample loading. Experiments were performed in triplicate with similar results. (c) PC12 cells were transiently transfected with plasmids encoding an IκBα-EGFP fusion protein or EGFP. After 24–48 h recovery, cells were treated with vehicle, NGF (1 ng/ml), or TNF-α (10 ng/ml). Cells overexpressing EGFP were exposed to TNF-α (EGFP + TNF-α). Quantification of changes in EGFP fluorescence after a 10-min exposure to vehicle, NGF, or TNF-α. Data are mean ± SEM from n = 4 separate transfection experiments per treatment. Data are given as change in average pixel intensities compared with the first image.
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Figure 4: TNF-α, but not NGF induces degradation of IκBs α and β. Degradation of IκBα and IκBβ in PC12 cells treated with (a) TNF-α (10 ng/ml) or (b) NGF (1 ng/ml) for the indicated period of time. 50 μg protein extract were separated on 12% SDS-PAGE, blotted onto nitrocellulose membrane, and IκBα or β was detected using rabbit polyclonal antibodies. Membranes were stripped and probed with an α-tubulin mouse monoclonal antibody as control for equal sample loading. Experiments were performed in triplicate with similar results. (c) PC12 cells were transiently transfected with plasmids encoding an IκBα-EGFP fusion protein or EGFP. After 24–48 h recovery, cells were treated with vehicle, NGF (1 ng/ml), or TNF-α (10 ng/ml). Cells overexpressing EGFP were exposed to TNF-α (EGFP + TNF-α). Quantification of changes in EGFP fluorescence after a 10-min exposure to vehicle, NGF, or TNF-α. Data are mean ± SEM from n = 4 separate transfection experiments per treatment. Data are given as change in average pixel intensities compared with the first image.

Mentions: IκB degradation has been shown to be required for activation of NFκB by proinflammatory cytokines (Palombella et al. 1994). We therefore determined a time course of IκBα protein degradation in PC12 cells exposed to TNF-α or NGF (Fig. 4, a and b). TNF-α induced significant IκBα degradation, starting 10 min after the onset of treatment. In contrast, treatment with NGF for up to 8 h failed to induce significant degradation of IκBα. NGF also failed to trigger the degradation of a second NFκB inhibitory protein, IκBβ (Thompson et al. 1995). In contrast, IκBβ degradation also occurred in TNF-α–treated cultures, albeit with slower kinetics.


Activation of nuclear factor kappaB and Bcl-x survival gene expression by nerve growth factor requires tyrosine phosphorylation of IkappaBalpha.

Bui NT, Livolsi A, Peyron JF, Prehn JH - J. Cell Biol. (2001)

TNF-α, but not NGF induces degradation of IκBs α and β. Degradation of IκBα and IκBβ in PC12 cells treated with (a) TNF-α (10 ng/ml) or (b) NGF (1 ng/ml) for the indicated period of time. 50 μg protein extract were separated on 12% SDS-PAGE, blotted onto nitrocellulose membrane, and IκBα or β was detected using rabbit polyclonal antibodies. Membranes were stripped and probed with an α-tubulin mouse monoclonal antibody as control for equal sample loading. Experiments were performed in triplicate with similar results. (c) PC12 cells were transiently transfected with plasmids encoding an IκBα-EGFP fusion protein or EGFP. After 24–48 h recovery, cells were treated with vehicle, NGF (1 ng/ml), or TNF-α (10 ng/ml). Cells overexpressing EGFP were exposed to TNF-α (EGFP + TNF-α). Quantification of changes in EGFP fluorescence after a 10-min exposure to vehicle, NGF, or TNF-α. Data are mean ± SEM from n = 4 separate transfection experiments per treatment. Data are given as change in average pixel intensities compared with the first image.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195773&req=5

Figure 4: TNF-α, but not NGF induces degradation of IκBs α and β. Degradation of IκBα and IκBβ in PC12 cells treated with (a) TNF-α (10 ng/ml) or (b) NGF (1 ng/ml) for the indicated period of time. 50 μg protein extract were separated on 12% SDS-PAGE, blotted onto nitrocellulose membrane, and IκBα or β was detected using rabbit polyclonal antibodies. Membranes were stripped and probed with an α-tubulin mouse monoclonal antibody as control for equal sample loading. Experiments were performed in triplicate with similar results. (c) PC12 cells were transiently transfected with plasmids encoding an IκBα-EGFP fusion protein or EGFP. After 24–48 h recovery, cells were treated with vehicle, NGF (1 ng/ml), or TNF-α (10 ng/ml). Cells overexpressing EGFP were exposed to TNF-α (EGFP + TNF-α). Quantification of changes in EGFP fluorescence after a 10-min exposure to vehicle, NGF, or TNF-α. Data are mean ± SEM from n = 4 separate transfection experiments per treatment. Data are given as change in average pixel intensities compared with the first image.
Mentions: IκB degradation has been shown to be required for activation of NFκB by proinflammatory cytokines (Palombella et al. 1994). We therefore determined a time course of IκBα protein degradation in PC12 cells exposed to TNF-α or NGF (Fig. 4, a and b). TNF-α induced significant IκBα degradation, starting 10 min after the onset of treatment. In contrast, treatment with NGF for up to 8 h failed to induce significant degradation of IκBα. NGF also failed to trigger the degradation of a second NFκB inhibitory protein, IκBβ (Thompson et al. 1995). In contrast, IκBβ degradation also occurred in TNF-α–treated cultures, albeit with slower kinetics.

Bottom Line: Moreover, in contrast to TNF-alpha, NGF failed to phosphorylate IkappaBalpha at serine residue 32, but instead caused significant tyrosine phosphorylation.Conversely, overexpression of a dominant negative mutant of TNF receptor-associated factor-6 blocked TNF-alpha-, but not NGF-induced NFkappaB activation.We conclude that NGF and TNF-alpha induce different signaling pathways in neurons to activate NFkappaB and bcl-x gene expression.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Center for Clinical Research, Research Group "Apoptosis and Cell Death,", D-48149 Münster, Germany.

ABSTRACT
NGF has been shown to support neuron survival by activating the transcription factor nuclear factor-kappaB (NFkappaB). We investigated the effect of NGF on the expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein. Treatment of rat pheochromocytoma PC12 cells, human neuroblastoma SH-SY5Y cells, or primary rat hippocampal neurons with NGF (0.1-10 ng/ml) increased the expression of bcl-xL mRNA and protein. Reporter gene analysis revealed a significant increase in NFkappaB activity after treatment with NGF that was associated with increased nuclear translocation of the active NFkappaB p65 subunit. NGF-induced NFkappaB activity and Bcl-xL expression were inhibited in cells overexpressing the NFkappaB inhibitor, IkappaBalpha. Unlike tumor necrosis factor-alpha (TNF-alpha), however, NGF-induced NFkappaB activation occurred without significant degradation of IkappaBs determined by Western blot analysis and time-lapse imaging of neurons expressing green fluorescent protein-tagged IkappaBalpha. Moreover, in contrast to TNF-alpha, NGF failed to phosphorylate IkappaBalpha at serine residue 32, but instead caused significant tyrosine phosphorylation. Overexpression of a Y42F mutant of IkappaBalpha potently suppressed NFG-, but not TNF-alpha-induced NFkappaB activation. Conversely, overexpression of a dominant negative mutant of TNF receptor-associated factor-6 blocked TNF-alpha-, but not NGF-induced NFkappaB activation. We conclude that NGF and TNF-alpha induce different signaling pathways in neurons to activate NFkappaB and bcl-x gene expression.

Show MeSH
Related in: MedlinePlus