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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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BIMEL expression promotes loss of mitochondrial cytochrome c but does not decrease survival in NGF-deprived neurons exposed to low O2. Neurons were infected with Ad-BIMEL/EGFP or Ad-EGFP adenoviruses (MOI = 100). The next day, NGF was withdrawn and the cells were immediately transferred to 1% O2. (A and B) After 24 h of NGF deprivation, the cells were fixed and analyzed for immunofluorescence using anti–cytochrome c antibody. The three panels for each treatment are of the same field of view. Bar, 15 μm. The percentage of EGFP-expressing, BIMEL/EGFP-expressing, and control (uninfected) cells showing punctate cytochrome c immunofluorescence was determined (means ± SEM, n = 3). Arrows in A point to a neuron expressing BIMEL-EGFP (top), its Hoechst-stained nucleus (middle), and its lack of punctate cytochrome c immunofluorescence (bottom). (C) Neurons were treated as described above except that NGF deprivation was continued for 48 h. The cells were then stained with Hoechst dye and scored for viability (mean ± SEM, n = 3). Except in the case of uninfected cells, only neurons positive for BIMEL/EGFP or EGFP expression were scored in B and C.
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fig5: BIMEL expression promotes loss of mitochondrial cytochrome c but does not decrease survival in NGF-deprived neurons exposed to low O2. Neurons were infected with Ad-BIMEL/EGFP or Ad-EGFP adenoviruses (MOI = 100). The next day, NGF was withdrawn and the cells were immediately transferred to 1% O2. (A and B) After 24 h of NGF deprivation, the cells were fixed and analyzed for immunofluorescence using anti–cytochrome c antibody. The three panels for each treatment are of the same field of view. Bar, 15 μm. The percentage of EGFP-expressing, BIMEL/EGFP-expressing, and control (uninfected) cells showing punctate cytochrome c immunofluorescence was determined (means ± SEM, n = 3). Arrows in A point to a neuron expressing BIMEL-EGFP (top), its Hoechst-stained nucleus (middle), and its lack of punctate cytochrome c immunofluorescence (bottom). (C) Neurons were treated as described above except that NGF deprivation was continued for 48 h. The cells were then stained with Hoechst dye and scored for viability (mean ± SEM, n = 3). Except in the case of uninfected cells, only neurons positive for BIMEL/EGFP or EGFP expression were scored in B and C.

Mentions: The results described above suggest a model in which low O2 inhibits cell death by suppressing BIMEL induction, which in turn inhibits the release of cytochrome c from mitochondria and ultimately cell death. As a test of this idea, we infected neurons with a control adenovirus expressing EGFP or one expressing a BIMEL/EGFP fusion protein. The next day, the cells were deprived of NGF and switched to a 1% O2 environment for an additional 24 h, after which the cells were examined by immunofluorescence for cytochrome c localization. Parallel cultures were deprived of NGF for 48 h to assess the effects of BIMEL expression on cell survival. The majority of control EGFP-expressing neurons exposed to low O2 during NGF deprivation retained intense, punctate cytochrome c immunofluorescence (Fig. 5, A and B), similar to the results shown with uninfected neurons in Fig. 2. In contrast, very few of the NGF-deprived neurons exposed to low O2 and expressing BIMEL/EGFP retained punctate cytochrome c labeling. Despite their apparent lack of mitochondrial-localized cytochrome c, the majority of cells expressing BIMEL/EGFP remained healthy 48 h after withdrawing NGF (Fig. 5 C). Thus, whereas forced expression of BIMEL can overcome the block to cytochrome c release, there must be additional processes induced by low O2 (apparently downstream or independent of cytochrome c release) that can sustain cell survival in the absence of NGF.


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)

BIMEL expression promotes loss of mitochondrial cytochrome c but does not decrease survival in NGF-deprived neurons exposed to low O2. Neurons were infected with Ad-BIMEL/EGFP or Ad-EGFP adenoviruses (MOI = 100). The next day, NGF was withdrawn and the cells were immediately transferred to 1% O2. (A and B) After 24 h of NGF deprivation, the cells were fixed and analyzed for immunofluorescence using anti–cytochrome c antibody. The three panels for each treatment are of the same field of view. Bar, 15 μm. The percentage of EGFP-expressing, BIMEL/EGFP-expressing, and control (uninfected) cells showing punctate cytochrome c immunofluorescence was determined (means ± SEM, n = 3). Arrows in A point to a neuron expressing BIMEL-EGFP (top), its Hoechst-stained nucleus (middle), and its lack of punctate cytochrome c immunofluorescence (bottom). (C) Neurons were treated as described above except that NGF deprivation was continued for 48 h. The cells were then stained with Hoechst dye and scored for viability (mean ± SEM, n = 3). Except in the case of uninfected cells, only neurons positive for BIMEL/EGFP or EGFP expression were scored in B and C.
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Related In: Results  -  Collection

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fig5: BIMEL expression promotes loss of mitochondrial cytochrome c but does not decrease survival in NGF-deprived neurons exposed to low O2. Neurons were infected with Ad-BIMEL/EGFP or Ad-EGFP adenoviruses (MOI = 100). The next day, NGF was withdrawn and the cells were immediately transferred to 1% O2. (A and B) After 24 h of NGF deprivation, the cells were fixed and analyzed for immunofluorescence using anti–cytochrome c antibody. The three panels for each treatment are of the same field of view. Bar, 15 μm. The percentage of EGFP-expressing, BIMEL/EGFP-expressing, and control (uninfected) cells showing punctate cytochrome c immunofluorescence was determined (means ± SEM, n = 3). Arrows in A point to a neuron expressing BIMEL-EGFP (top), its Hoechst-stained nucleus (middle), and its lack of punctate cytochrome c immunofluorescence (bottom). (C) Neurons were treated as described above except that NGF deprivation was continued for 48 h. The cells were then stained with Hoechst dye and scored for viability (mean ± SEM, n = 3). Except in the case of uninfected cells, only neurons positive for BIMEL/EGFP or EGFP expression were scored in B and C.
Mentions: The results described above suggest a model in which low O2 inhibits cell death by suppressing BIMEL induction, which in turn inhibits the release of cytochrome c from mitochondria and ultimately cell death. As a test of this idea, we infected neurons with a control adenovirus expressing EGFP or one expressing a BIMEL/EGFP fusion protein. The next day, the cells were deprived of NGF and switched to a 1% O2 environment for an additional 24 h, after which the cells were examined by immunofluorescence for cytochrome c localization. Parallel cultures were deprived of NGF for 48 h to assess the effects of BIMEL expression on cell survival. The majority of control EGFP-expressing neurons exposed to low O2 during NGF deprivation retained intense, punctate cytochrome c immunofluorescence (Fig. 5, A and B), similar to the results shown with uninfected neurons in Fig. 2. In contrast, very few of the NGF-deprived neurons exposed to low O2 and expressing BIMEL/EGFP retained punctate cytochrome c labeling. Despite their apparent lack of mitochondrial-localized cytochrome c, the majority of cells expressing BIMEL/EGFP remained healthy 48 h after withdrawing NGF (Fig. 5 C). Thus, whereas forced expression of BIMEL can overcome the block to cytochrome c release, there must be additional processes induced by low O2 (apparently downstream or independent of cytochrome c release) that can sustain cell survival in the absence of NGF.

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