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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 affects only some of the pathways involved in the induction of BIMEL after NGF withdrawal. (A) Neurons were treated with or without NGF and exposed to 20% or 1% O2 for the indicated times. Whole cell lysates were prepared and immunoblotted using anti–phospho-ERK1/2 or anti-ERK1/2 antibody. (B) Neurons were kept in the presence of NGF or deprived of NGF for 20 h at either 20% or 1% O2. Proteasome inhibitor (MG132, 50 μM) was added to one set of the NGF-deprived cultures. Whole cell lysates were immunoblotted with anti-BIM and anti-actin antibodies. The concentration of MG132 used was sufficient to inhibit proteasome activity as evidenced by its ability to stabilize HIF-1α (not depicted). (C) mRNA levels for BIMEL and cyclophilin were analyzed by semi-quantitative RT-PCR using total RNA prepared from neurons treated in the presence or absence of NGF for 20 h at 20% or 1% O2. BIMEL mRNA levels increased 4.2-fold during NGF deprivation in neurons exposed to 20% O2 compared with 1.9-fold in neurons exposed to 1% O2 (normalized to cyclophilin, n = 2). (D) Whole cell lysates were prepared from neurons treated with or without NGF and exposed to 1% O2 for the indicated times. The lysates were then immunoblotted with anti–phospho-c-Jun (Ser-63) and anti–phospho-Akt (Ser-473) antibodies. Arrows indicate the positions of the phosphorylated proteins. (E) Whole cell extracts prepared from cells treated in the presence or absence of NGF for 20 h at 20% or 1% O2 were immunoblotted with anti–phospho-FKHRL1 (Thr-32), anti–phospho-Akt, and anti–Akt antibodies. Similar results were seen in at least three independent experiments.
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fig4: Low O2 affects only some of the pathways involved in the induction of BIMEL after NGF withdrawal. (A) Neurons were treated with or without NGF and exposed to 20% or 1% O2 for the indicated times. Whole cell lysates were prepared and immunoblotted using anti–phospho-ERK1/2 or anti-ERK1/2 antibody. (B) Neurons were kept in the presence of NGF or deprived of NGF for 20 h at either 20% or 1% O2. Proteasome inhibitor (MG132, 50 μM) was added to one set of the NGF-deprived cultures. Whole cell lysates were immunoblotted with anti-BIM and anti-actin antibodies. The concentration of MG132 used was sufficient to inhibit proteasome activity as evidenced by its ability to stabilize HIF-1α (not depicted). (C) mRNA levels for BIMEL and cyclophilin were analyzed by semi-quantitative RT-PCR using total RNA prepared from neurons treated in the presence or absence of NGF for 20 h at 20% or 1% O2. BIMEL mRNA levels increased 4.2-fold during NGF deprivation in neurons exposed to 20% O2 compared with 1.9-fold in neurons exposed to 1% O2 (normalized to cyclophilin, n = 2). (D) Whole cell lysates were prepared from neurons treated with or without NGF and exposed to 1% O2 for the indicated times. The lysates were then immunoblotted with anti–phospho-c-Jun (Ser-63) and anti–phospho-Akt (Ser-473) antibodies. Arrows indicate the positions of the phosphorylated proteins. (E) Whole cell extracts prepared from cells treated in the presence or absence of NGF for 20 h at 20% or 1% O2 were immunoblotted with anti–phospho-FKHRL1 (Thr-32), anti–phospho-Akt, and anti–Akt antibodies. Similar results were seen in at least three independent experiments.

Mentions: Recent studies have suggested that phosphorylation of BIMEL by extracellular signal-regulated kinases (ERKs) is involved in targeting BIMEL to the proteasome (Biswas and Greene, 2002; Ley et al., 2003). We considered the possibility that a similar mechanism might underlie the suppression of BIMEL expression by low O2. Exposing control NGF-maintained neurons to 1% O2 for 20 h resulted in a small increase in phosphorylated ERK1/2 (Fig. 4 A, top). Nonetheless, phosphorylated ERK1/2 levels decreased similarly during NGF deprivation regardless of O2 tension (Fig. 4 A), suggesting that reducing O2 was not sufficient to stimulate ERK activation in sympathetic neurons in the absence of NGF signaling.


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 affects only some of the pathways involved in the induction of BIMEL after NGF withdrawal. (A) Neurons were treated with or without NGF and exposed to 20% or 1% O2 for the indicated times. Whole cell lysates were prepared and immunoblotted using anti–phospho-ERK1/2 or anti-ERK1/2 antibody. (B) Neurons were kept in the presence of NGF or deprived of NGF for 20 h at either 20% or 1% O2. Proteasome inhibitor (MG132, 50 μM) was added to one set of the NGF-deprived cultures. Whole cell lysates were immunoblotted with anti-BIM and anti-actin antibodies. The concentration of MG132 used was sufficient to inhibit proteasome activity as evidenced by its ability to stabilize HIF-1α (not depicted). (C) mRNA levels for BIMEL and cyclophilin were analyzed by semi-quantitative RT-PCR using total RNA prepared from neurons treated in the presence or absence of NGF for 20 h at 20% or 1% O2. BIMEL mRNA levels increased 4.2-fold during NGF deprivation in neurons exposed to 20% O2 compared with 1.9-fold in neurons exposed to 1% O2 (normalized to cyclophilin, n = 2). (D) Whole cell lysates were prepared from neurons treated with or without NGF and exposed to 1% O2 for the indicated times. The lysates were then immunoblotted with anti–phospho-c-Jun (Ser-63) and anti–phospho-Akt (Ser-473) antibodies. Arrows indicate the positions of the phosphorylated proteins. (E) Whole cell extracts prepared from cells treated in the presence or absence of NGF for 20 h at 20% or 1% O2 were immunoblotted with anti–phospho-FKHRL1 (Thr-32), anti–phospho-Akt, and anti–Akt antibodies. Similar results were seen in at least three independent experiments.
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Related In: Results  -  Collection

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fig4: Low O2 affects only some of the pathways involved in the induction of BIMEL after NGF withdrawal. (A) Neurons were treated with or without NGF and exposed to 20% or 1% O2 for the indicated times. Whole cell lysates were prepared and immunoblotted using anti–phospho-ERK1/2 or anti-ERK1/2 antibody. (B) Neurons were kept in the presence of NGF or deprived of NGF for 20 h at either 20% or 1% O2. Proteasome inhibitor (MG132, 50 μM) was added to one set of the NGF-deprived cultures. Whole cell lysates were immunoblotted with anti-BIM and anti-actin antibodies. The concentration of MG132 used was sufficient to inhibit proteasome activity as evidenced by its ability to stabilize HIF-1α (not depicted). (C) mRNA levels for BIMEL and cyclophilin were analyzed by semi-quantitative RT-PCR using total RNA prepared from neurons treated in the presence or absence of NGF for 20 h at 20% or 1% O2. BIMEL mRNA levels increased 4.2-fold during NGF deprivation in neurons exposed to 20% O2 compared with 1.9-fold in neurons exposed to 1% O2 (normalized to cyclophilin, n = 2). (D) Whole cell lysates were prepared from neurons treated with or without NGF and exposed to 1% O2 for the indicated times. The lysates were then immunoblotted with anti–phospho-c-Jun (Ser-63) and anti–phospho-Akt (Ser-473) antibodies. Arrows indicate the positions of the phosphorylated proteins. (E) Whole cell extracts prepared from cells treated in the presence or absence of NGF for 20 h at 20% or 1% O2 were immunoblotted with anti–phospho-FKHRL1 (Thr-32), anti–phospho-Akt, and anti–Akt antibodies. Similar results were seen in at least three independent experiments.
Mentions: Recent studies have suggested that phosphorylation of BIMEL by extracellular signal-regulated kinases (ERKs) is involved in targeting BIMEL to the proteasome (Biswas and Greene, 2002; Ley et al., 2003). We considered the possibility that a similar mechanism might underlie the suppression of BIMEL expression by low O2. Exposing control NGF-maintained neurons to 1% O2 for 20 h resulted in a small increase in phosphorylated ERK1/2 (Fig. 4 A, top). Nonetheless, phosphorylated ERK1/2 levels decreased similarly during NGF deprivation regardless of O2 tension (Fig. 4 A), suggesting that reducing O2 was not sufficient to stimulate ERK activation in sympathetic neurons in the absence of NGF signaling.

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