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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 inhibits the loss of cytochrome c from mitochondria. Neurons were deprived of NGF or refed with fresh NGF-containing media and cultured at 20% or 1% O2 for 24 h. (A and B) Immunofluorescence was performed using an anti–cytochrome c antibody and cells were counterstained with Hoechst 33,342. Phase-contrast and epifluorescence images show a single field of view for each condition. Panels 1–4, 20% O2; panels 5–8, 1% O2. Bar, 20 μm. The percentage of cells with punctate cytochrome c (mean ± SEM) was determined in three independent experiments. (C) Whole cell lysates from neurons treated as described above were separated by 15% SDS-PAGE and immunoblotted with anti–cytochrome c antibody. NS represents a nonspecific protein detected by the primary antibody (similar levels of this protein confirm equal loading of the gel). NGF deprivation resulted in a 73 ± 9% decrease in cytochrome c levels in neurons exposed to 20% O2 compared with a 30 ± 3% decrease in neurons exposed to 1% O2 (percent decreases are the mean ± SEM from three independent experiments).
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fig2: Low O2 inhibits the loss of cytochrome c from mitochondria. Neurons were deprived of NGF or refed with fresh NGF-containing media and cultured at 20% or 1% O2 for 24 h. (A and B) Immunofluorescence was performed using an anti–cytochrome c antibody and cells were counterstained with Hoechst 33,342. Phase-contrast and epifluorescence images show a single field of view for each condition. Panels 1–4, 20% O2; panels 5–8, 1% O2. Bar, 20 μm. The percentage of cells with punctate cytochrome c (mean ± SEM) was determined in three independent experiments. (C) Whole cell lysates from neurons treated as described above were separated by 15% SDS-PAGE and immunoblotted with anti–cytochrome c antibody. NS represents a nonspecific protein detected by the primary antibody (similar levels of this protein confirm equal loading of the gel). NGF deprivation resulted in a 73 ± 9% decrease in cytochrome c levels in neurons exposed to 20% O2 compared with a 30 ± 3% decrease in neurons exposed to 1% O2 (percent decreases are the mean ± SEM from three independent experiments).

Mentions: Using immunofluorescence, we investigated the effect of low O2 on the localization of cytochrome c after NGF withdrawal. Neurons maintained with NGF at 20% O2 have a punctate, cytoplasmic distribution of cytochrome c immunofluorescence, indicative of its mitochondrial localization (Fig. 2, A and B). As seen by others (Deshmukh and Johnson, 1998; Neame et al., 1998), withdrawing NGF from these neurons results in an almost complete loss of punctate cytochrome c immunofluorescence. In contrast, the majority of neurons exposed to 1% O2 during NGF deprivation retained punctate cytochrome c immunofluorescence similar to NGF-maintained neurons.


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 inhibits the loss of cytochrome c from mitochondria. Neurons were deprived of NGF or refed with fresh NGF-containing media and cultured at 20% or 1% O2 for 24 h. (A and B) Immunofluorescence was performed using an anti–cytochrome c antibody and cells were counterstained with Hoechst 33,342. Phase-contrast and epifluorescence images show a single field of view for each condition. Panels 1–4, 20% O2; panels 5–8, 1% O2. Bar, 20 μm. The percentage of cells with punctate cytochrome c (mean ± SEM) was determined in three independent experiments. (C) Whole cell lysates from neurons treated as described above were separated by 15% SDS-PAGE and immunoblotted with anti–cytochrome c antibody. NS represents a nonspecific protein detected by the primary antibody (similar levels of this protein confirm equal loading of the gel). NGF deprivation resulted in a 73 ± 9% decrease in cytochrome c levels in neurons exposed to 20% O2 compared with a 30 ± 3% decrease in neurons exposed to 1% O2 (percent decreases are the mean ± SEM from three independent experiments).
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Related In: Results  -  Collection

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fig2: Low O2 inhibits the loss of cytochrome c from mitochondria. Neurons were deprived of NGF or refed with fresh NGF-containing media and cultured at 20% or 1% O2 for 24 h. (A and B) Immunofluorescence was performed using an anti–cytochrome c antibody and cells were counterstained with Hoechst 33,342. Phase-contrast and epifluorescence images show a single field of view for each condition. Panels 1–4, 20% O2; panels 5–8, 1% O2. Bar, 20 μm. The percentage of cells with punctate cytochrome c (mean ± SEM) was determined in three independent experiments. (C) Whole cell lysates from neurons treated as described above were separated by 15% SDS-PAGE and immunoblotted with anti–cytochrome c antibody. NS represents a nonspecific protein detected by the primary antibody (similar levels of this protein confirm equal loading of the gel). NGF deprivation resulted in a 73 ± 9% decrease in cytochrome c levels in neurons exposed to 20% O2 compared with a 30 ± 3% decrease in neurons exposed to 1% O2 (percent decreases are the mean ± SEM from three independent experiments).
Mentions: Using immunofluorescence, we investigated the effect of low O2 on the localization of cytochrome c after NGF withdrawal. Neurons maintained with NGF at 20% O2 have a punctate, cytoplasmic distribution of cytochrome c immunofluorescence, indicative of its mitochondrial localization (Fig. 2, A and B). As seen by others (Deshmukh and Johnson, 1998; Neame et al., 1998), withdrawing NGF from these neurons results in an almost complete loss of punctate cytochrome c immunofluorescence. In contrast, the majority of neurons exposed to 1% O2 during NGF deprivation retained punctate cytochrome c immunofluorescence similar to NGF-maintained neurons.

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