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Inhibition of NGF deprivation-induced death by low oxygen involves suppression of BIMEL and activation of HIF-1.

Xie L, Johnson RS, Freeman RS - J. Cell Biol. (2005)

Bottom Line: Forced BIM(EL) expression removed the block to cytochrome c release but did not prevent protection by low O(2).Exposing neurons to low O(2) also activated hypoxia-inducible factor (HIF) and expression of a stabilized form of HIF-1alpha (HIF-1alpha(PP-->AG)) inhibited cell death in normoxic, NGF-deprived cells.Targeted deletion of HIF-1alpha partially suppressed the protective effect of low O(2), whereas deletion of HIF-1alpha combined with forced BIM(EL) expression completely reversed the ability of low O(2) to inhibit cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, NY 14642, USA.

ABSTRACT
Changes in O(2) tension can significantly impact cell survival, yet the mechanisms underlying these effects are not well understood. Here, we report that maintaining sympathetic neurons under low O(2) inhibits apoptosis caused by NGF deprivation. Low O(2) exposure blocked cytochrome c release after NGF withdrawal, in part by suppressing the up-regulation of BIM(EL). Forced BIM(EL) expression removed the block to cytochrome c release but did not prevent protection by low O(2). Exposing neurons to low O(2) also activated hypoxia-inducible factor (HIF) and expression of a stabilized form of HIF-1alpha (HIF-1alpha(PP-->AG)) inhibited cell death in normoxic, NGF-deprived cells. Targeted deletion of HIF-1alpha partially suppressed the protective effect of low O(2), whereas deletion of HIF-1alpha combined with forced BIM(EL) expression completely reversed the ability of low O(2) to inhibit cell death. These data suggest a new model for how O(2) tension can influence apoptotic events that underlie trophic factor deprivation-induced cell death.

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Low O2 exposure activates HIF in the presence or absence of NGF. (A) Neurons were infected with Ad-HRE-luciferase (MOI = 10) and 48 h later either continued in the presence of NGF or deprived of NGF for an additional 12 h at 20% or 1% O2. Cells were then lysed and relative luciferase activities were determined (mean ± SEM of triplicate wells; similar results were obtained in a second independent experiment). (B) Neurons were either deprived of NGF and incubated at 1% O2 or kept in the presence of NGF and cultured at 20% O2. After 20 h, HIF-1α protein expression was analyzed by immunoprecipitation followed by immunoblotting with an anti–HIF-1α antibody.
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fig6: Low O2 exposure activates HIF in the presence or absence of NGF. (A) Neurons were infected with Ad-HRE-luciferase (MOI = 10) and 48 h later either continued in the presence of NGF or deprived of NGF for an additional 12 h at 20% or 1% O2. Cells were then lysed and relative luciferase activities were determined (mean ± SEM of triplicate wells; similar results were obtained in a second independent experiment). (B) Neurons were either deprived of NGF and incubated at 1% O2 or kept in the presence of NGF and cultured at 20% O2. After 20 h, HIF-1α protein expression was analyzed by immunoprecipitation followed by immunoblotting with an anti–HIF-1α antibody.

Mentions: Low O2 exposure results in increased HIF activity. Because BIMEL expression failed to reverse the neuroprotective effect of low O2, we wondered if activation of HIF might contribute to the enhanced survival seen under these conditions. In initial experiments, we infected neurons using an adenovirus that expresses a luciferase reporter gene under the control of four tandem hypoxia response elements (Ad-HRE-luciferase). Not surprisingly, NGF-maintained neurons exposed to 1% O2 had increased HIF reporter gene activity compared with neurons kept under standard cell culture conditions (Fig. 6 A). NGF-deprived neurons exposed to 1% O2 displayed a similar elevation in HRE-luciferase activity, demonstrating that low O2-mediated activation of HIF in these neurons can occur in the absence of NGF signaling. HIF-1α protein levels also increased under the same conditions suggesting that at least part of the increase in HIF activity is due to HIF-1 (Fig. 6 B).


Inhibition of NGF deprivation-induced death by low oxygen involves suppression of BIMEL and activation of HIF-1.

Xie L, Johnson RS, Freeman RS - J. Cell Biol. (2005)

Low O2 exposure activates HIF in the presence or absence of NGF. (A) Neurons were infected with Ad-HRE-luciferase (MOI = 10) and 48 h later either continued in the presence of NGF or deprived of NGF for an additional 12 h at 20% or 1% O2. Cells were then lysed and relative luciferase activities were determined (mean ± SEM of triplicate wells; similar results were obtained in a second independent experiment). (B) Neurons were either deprived of NGF and incubated at 1% O2 or kept in the presence of NGF and cultured at 20% O2. After 20 h, HIF-1α protein expression was analyzed by immunoprecipitation followed by immunoblotting with an anti–HIF-1α antibody.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171791&req=5

fig6: Low O2 exposure activates HIF in the presence or absence of NGF. (A) Neurons were infected with Ad-HRE-luciferase (MOI = 10) and 48 h later either continued in the presence of NGF or deprived of NGF for an additional 12 h at 20% or 1% O2. Cells were then lysed and relative luciferase activities were determined (mean ± SEM of triplicate wells; similar results were obtained in a second independent experiment). (B) Neurons were either deprived of NGF and incubated at 1% O2 or kept in the presence of NGF and cultured at 20% O2. After 20 h, HIF-1α protein expression was analyzed by immunoprecipitation followed by immunoblotting with an anti–HIF-1α antibody.
Mentions: Low O2 exposure results in increased HIF activity. Because BIMEL expression failed to reverse the neuroprotective effect of low O2, we wondered if activation of HIF might contribute to the enhanced survival seen under these conditions. In initial experiments, we infected neurons using an adenovirus that expresses a luciferase reporter gene under the control of four tandem hypoxia response elements (Ad-HRE-luciferase). Not surprisingly, NGF-maintained neurons exposed to 1% O2 had increased HIF reporter gene activity compared with neurons kept under standard cell culture conditions (Fig. 6 A). NGF-deprived neurons exposed to 1% O2 displayed a similar elevation in HRE-luciferase activity, demonstrating that low O2-mediated activation of HIF in these neurons can occur in the absence of NGF signaling. HIF-1α protein levels also increased under the same conditions suggesting that at least part of the increase in HIF activity is due to HIF-1 (Fig. 6 B).

Bottom Line: Forced BIM(EL) expression removed the block to cytochrome c release but did not prevent protection by low O(2).Exposing neurons to low O(2) also activated hypoxia-inducible factor (HIF) and expression of a stabilized form of HIF-1alpha (HIF-1alpha(PP-->AG)) inhibited cell death in normoxic, NGF-deprived cells.Targeted deletion of HIF-1alpha partially suppressed the protective effect of low O(2), whereas deletion of HIF-1alpha combined with forced BIM(EL) expression completely reversed the ability of low O(2) to inhibit cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, NY 14642, USA.

ABSTRACT
Changes in O(2) tension can significantly impact cell survival, yet the mechanisms underlying these effects are not well understood. Here, we report that maintaining sympathetic neurons under low O(2) inhibits apoptosis caused by NGF deprivation. Low O(2) exposure blocked cytochrome c release after NGF withdrawal, in part by suppressing the up-regulation of BIM(EL). Forced BIM(EL) expression removed the block to cytochrome c release but did not prevent protection by low O(2). Exposing neurons to low O(2) also activated hypoxia-inducible factor (HIF) and expression of a stabilized form of HIF-1alpha (HIF-1alpha(PP-->AG)) inhibited cell death in normoxic, NGF-deprived cells. Targeted deletion of HIF-1alpha partially suppressed the protective effect of low O(2), whereas deletion of HIF-1alpha combined with forced BIM(EL) expression completely reversed the ability of low O(2) to inhibit cell death. These data suggest a new model for how O(2) tension can influence apoptotic events that underlie trophic factor deprivation-induced cell death.

Show MeSH
Related in: MedlinePlus