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Decreased apoptosome activity with neuronal differentiation sets the threshold for strict IAP regulation of apoptosis.

Wright KM, Linhoff MW, Potts PR, Deshmukh M - J. Cell Biol. (2004)

Bottom Line: We report that the ability of endogenous IAPs to effectively regulate caspase activation depends on the differentiation state of the cell.Neuronal differentiation was also accompanied with a marked reduction in Apaf-1, resulting in a significant decrease in apoptosome activity.Importantly, this decrease in Apaf-1 protein was directly linked to the increased ability of IAPs to stringently regulate apoptosis in neuronally differentiated PC12 and primary cells.

View Article: PubMed Central - PubMed

Affiliation: Curriculum in Neurobiology, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Despite the potential of the inhibitor of apoptosis proteins (IAPs) to block cytochrome c-dependent caspase activation, the critical function of IAPs in regulating mammalian apoptosis remains unclear. We report that the ability of endogenous IAPs to effectively regulate caspase activation depends on the differentiation state of the cell. Despite being expressed at equivalent levels, endogenous IAPs afforded no protection against cytochrome c-induced apoptosis in naive pheochromocytoma (PC12) cells, but were remarkably effective in doing so in neuronally differentiated cells. Neuronal differentiation was also accompanied with a marked reduction in Apaf-1, resulting in a significant decrease in apoptosome activity. Importantly, this decrease in Apaf-1 protein was directly linked to the increased ability of IAPs to stringently regulate apoptosis in neuronally differentiated PC12 and primary cells. These data illustrate specifically how the apoptotic pathway acquires increased regulation with cellular differentiation, and are the first to show that IAP function and apoptosome activity are coupled in cells.

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A marked reduction in Apaf-1 levels causes the reduced apoptosome activity in neuronally differentiated cells. (A) Western blots comparing the levels of Apaf-1, caspase-9, and lactate dehydrogenase (LDH) in naïve and neuronally differentiated cell lysates. Quantitation of protein levels (mean ± SEM of three independent experiments) for Apaf-1 and caspase-9 are shown. (B) The ability of exogenously added cytochrome c (10 μM) to activate caspases was examined in cytosolic lysates of neuronally differentiated PC12 cells alone (150 μg) or neuronally differentiated cell lysates (75 μg) mixed with cytosolic lysates from either wild-type, caspase-9−/−, or Apaf-1−/− fibroblasts (75 μg). (C) Cytosolic lysate (150 μg) from neuronally differentiated cells was incubated with cytochrome c alone (10 μM) or cytochrome c with Apaf-1 protein (10 nM) and assessed for caspase activation. Western analysis of these lysates probed for caspase-9 are shown below. (D) Neuronally differentiated cells were injected with plasmid DNA encoding EGFP (50 ng/μl) and plasmid DNA for vector alone, Apaf-1, or caspase-9 (200 ng/μl). After 24 h for DNA expression, GFP-positive cells were injected with cytochrome c (10 mg/ml) or rhodamine dextran and assessed for viability at the indicated times after the injections. Data shown are mean ± SEM of three independent experiments.
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fig5: A marked reduction in Apaf-1 levels causes the reduced apoptosome activity in neuronally differentiated cells. (A) Western blots comparing the levels of Apaf-1, caspase-9, and lactate dehydrogenase (LDH) in naïve and neuronally differentiated cell lysates. Quantitation of protein levels (mean ± SEM of three independent experiments) for Apaf-1 and caspase-9 are shown. (B) The ability of exogenously added cytochrome c (10 μM) to activate caspases was examined in cytosolic lysates of neuronally differentiated PC12 cells alone (150 μg) or neuronally differentiated cell lysates (75 μg) mixed with cytosolic lysates from either wild-type, caspase-9−/−, or Apaf-1−/− fibroblasts (75 μg). (C) Cytosolic lysate (150 μg) from neuronally differentiated cells was incubated with cytochrome c alone (10 μM) or cytochrome c with Apaf-1 protein (10 nM) and assessed for caspase activation. Western analysis of these lysates probed for caspase-9 are shown below. (D) Neuronally differentiated cells were injected with plasmid DNA encoding EGFP (50 ng/μl) and plasmid DNA for vector alone, Apaf-1, or caspase-9 (200 ng/μl). After 24 h for DNA expression, GFP-positive cells were injected with cytochrome c (10 mg/ml) or rhodamine dextran and assessed for viability at the indicated times after the injections. Data shown are mean ± SEM of three independent experiments.

Mentions: Next, we examined whether the reduced apoptosome activity in neuronally differentiated cells was due to limiting levels of Apaf-1 or procaspase-9. We found that levels of Apaf-1, but not caspase-9, were reduced by a striking 50% when naïve cells were neuronally differentiated for 12 d (Fig. 5 A).


Decreased apoptosome activity with neuronal differentiation sets the threshold for strict IAP regulation of apoptosis.

Wright KM, Linhoff MW, Potts PR, Deshmukh M - J. Cell Biol. (2004)

A marked reduction in Apaf-1 levels causes the reduced apoptosome activity in neuronally differentiated cells. (A) Western blots comparing the levels of Apaf-1, caspase-9, and lactate dehydrogenase (LDH) in naïve and neuronally differentiated cell lysates. Quantitation of protein levels (mean ± SEM of three independent experiments) for Apaf-1 and caspase-9 are shown. (B) The ability of exogenously added cytochrome c (10 μM) to activate caspases was examined in cytosolic lysates of neuronally differentiated PC12 cells alone (150 μg) or neuronally differentiated cell lysates (75 μg) mixed with cytosolic lysates from either wild-type, caspase-9−/−, or Apaf-1−/− fibroblasts (75 μg). (C) Cytosolic lysate (150 μg) from neuronally differentiated cells was incubated with cytochrome c alone (10 μM) or cytochrome c with Apaf-1 protein (10 nM) and assessed for caspase activation. Western analysis of these lysates probed for caspase-9 are shown below. (D) Neuronally differentiated cells were injected with plasmid DNA encoding EGFP (50 ng/μl) and plasmid DNA for vector alone, Apaf-1, or caspase-9 (200 ng/μl). After 24 h for DNA expression, GFP-positive cells were injected with cytochrome c (10 mg/ml) or rhodamine dextran and assessed for viability at the indicated times after the injections. Data shown are mean ± SEM of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172554&req=5

fig5: A marked reduction in Apaf-1 levels causes the reduced apoptosome activity in neuronally differentiated cells. (A) Western blots comparing the levels of Apaf-1, caspase-9, and lactate dehydrogenase (LDH) in naïve and neuronally differentiated cell lysates. Quantitation of protein levels (mean ± SEM of three independent experiments) for Apaf-1 and caspase-9 are shown. (B) The ability of exogenously added cytochrome c (10 μM) to activate caspases was examined in cytosolic lysates of neuronally differentiated PC12 cells alone (150 μg) or neuronally differentiated cell lysates (75 μg) mixed with cytosolic lysates from either wild-type, caspase-9−/−, or Apaf-1−/− fibroblasts (75 μg). (C) Cytosolic lysate (150 μg) from neuronally differentiated cells was incubated with cytochrome c alone (10 μM) or cytochrome c with Apaf-1 protein (10 nM) and assessed for caspase activation. Western analysis of these lysates probed for caspase-9 are shown below. (D) Neuronally differentiated cells were injected with plasmid DNA encoding EGFP (50 ng/μl) and plasmid DNA for vector alone, Apaf-1, or caspase-9 (200 ng/μl). After 24 h for DNA expression, GFP-positive cells were injected with cytochrome c (10 mg/ml) or rhodamine dextran and assessed for viability at the indicated times after the injections. Data shown are mean ± SEM of three independent experiments.
Mentions: Next, we examined whether the reduced apoptosome activity in neuronally differentiated cells was due to limiting levels of Apaf-1 or procaspase-9. We found that levels of Apaf-1, but not caspase-9, were reduced by a striking 50% when naïve cells were neuronally differentiated for 12 d (Fig. 5 A).

Bottom Line: We report that the ability of endogenous IAPs to effectively regulate caspase activation depends on the differentiation state of the cell.Neuronal differentiation was also accompanied with a marked reduction in Apaf-1, resulting in a significant decrease in apoptosome activity.Importantly, this decrease in Apaf-1 protein was directly linked to the increased ability of IAPs to stringently regulate apoptosis in neuronally differentiated PC12 and primary cells.

View Article: PubMed Central - PubMed

Affiliation: Curriculum in Neurobiology, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Despite the potential of the inhibitor of apoptosis proteins (IAPs) to block cytochrome c-dependent caspase activation, the critical function of IAPs in regulating mammalian apoptosis remains unclear. We report that the ability of endogenous IAPs to effectively regulate caspase activation depends on the differentiation state of the cell. Despite being expressed at equivalent levels, endogenous IAPs afforded no protection against cytochrome c-induced apoptosis in naive pheochromocytoma (PC12) cells, but were remarkably effective in doing so in neuronally differentiated cells. Neuronal differentiation was also accompanied with a marked reduction in Apaf-1, resulting in a significant decrease in apoptosome activity. Importantly, this decrease in Apaf-1 protein was directly linked to the increased ability of IAPs to stringently regulate apoptosis in neuronally differentiated PC12 and primary cells. These data illustrate specifically how the apoptotic pathway acquires increased regulation with cellular differentiation, and are the first to show that IAP function and apoptosome activity are coupled in cells.

Show MeSH
Related in: MedlinePlus