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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 induction during NGF deprivation is inhibited by exposure to low O2. (A) Neurons were treated as indicated and after 20 h whole cell lysates were prepared and immunoblotted with anti–phospho-c-Jun (Ser-63), anti BCL-XL, anti-BAX, anti-BIM, and anti-actin antibodies. (B) BIMEL protein levels were quantified using immunoblots from three independent experiments. The results (mean fold increase ± SEM) represent the ratio of BIMEL protein in NGF-deprived neurons to that in NGF-maintained neurons at either 20% or 1% O2.
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fig3: BIMEL induction during NGF deprivation is inhibited by exposure to low O2. (A) Neurons were treated as indicated and after 20 h whole cell lysates were prepared and immunoblotted with anti–phospho-c-Jun (Ser-63), anti BCL-XL, anti-BAX, anti-BIM, and anti-actin antibodies. (B) BIMEL protein levels were quantified using immunoblots from three independent experiments. The results (mean fold increase ± SEM) represent the ratio of BIMEL protein in NGF-deprived neurons to that in NGF-maintained neurons at either 20% or 1% O2.

Mentions: Because the BCL-2 family proteins BAX and BIMEL are critical for cytochrome c release after NGF withdrawal, we compared their protein levels before and after NGF withdrawal in neurons cultured under standard and reduced O2. We also analyzed expression of the pro-survival protein BCL-XL and for changes in the phosphorylation of c-Jun. Phosphorylation of c-Jun increases in neurons deprived of NGF under standard culture conditions (Ham et al., 1995; Virdee et al., 1997) and we observed an essentially identical increase in neurons exposed to low O2 (Fig. 3 A). BAX and BCL-XL protein levels remained unchanged after 20 h of NGF withdrawal, regardless of O2 tension. BIMEL protein levels, on the other hand, increased an average of fourfold when neurons were deprived of NGF under standard O2 conditions; but when NGF deprivation was performed at 1% O2, a much smaller increase in BIMEL was observed (Fig. 3, A and B; also see Fig. 4 B). This effect appeared to be specific because low O2 did not inhibit c-Jun phosphorylation or decrease the expression of any of the other proteins examined.


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 induction during NGF deprivation is inhibited by exposure to low O2. (A) Neurons were treated as indicated and after 20 h whole cell lysates were prepared and immunoblotted with anti–phospho-c-Jun (Ser-63), anti BCL-XL, anti-BAX, anti-BIM, and anti-actin antibodies. (B) BIMEL protein levels were quantified using immunoblots from three independent experiments. The results (mean fold increase ± SEM) represent the ratio of BIMEL protein in NGF-deprived neurons to that in NGF-maintained neurons at either 20% or 1% O2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171791&req=5

fig3: BIMEL induction during NGF deprivation is inhibited by exposure to low O2. (A) Neurons were treated as indicated and after 20 h whole cell lysates were prepared and immunoblotted with anti–phospho-c-Jun (Ser-63), anti BCL-XL, anti-BAX, anti-BIM, and anti-actin antibodies. (B) BIMEL protein levels were quantified using immunoblots from three independent experiments. The results (mean fold increase ± SEM) represent the ratio of BIMEL protein in NGF-deprived neurons to that in NGF-maintained neurons at either 20% or 1% O2.
Mentions: Because the BCL-2 family proteins BAX and BIMEL are critical for cytochrome c release after NGF withdrawal, we compared their protein levels before and after NGF withdrawal in neurons cultured under standard and reduced O2. We also analyzed expression of the pro-survival protein BCL-XL and for changes in the phosphorylation of c-Jun. Phosphorylation of c-Jun increases in neurons deprived of NGF under standard culture conditions (Ham et al., 1995; Virdee et al., 1997) and we observed an essentially identical increase in neurons exposed to low O2 (Fig. 3 A). BAX and BCL-XL protein levels remained unchanged after 20 h of NGF withdrawal, regardless of O2 tension. BIMEL protein levels, on the other hand, increased an average of fourfold when neurons were deprived of NGF under standard O2 conditions; but when NGF deprivation was performed at 1% O2, a much smaller increase in BIMEL was observed (Fig. 3, A and B; also see Fig. 4 B). This effect appeared to be specific because low O2 did not inhibit c-Jun phosphorylation or decrease the expression of any of the other proteins examined.

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