Limits...
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.

Show MeSH

Related in: MedlinePlus

Reducing the apoptosome efficiency with addition of suboptimum cytochrome c permits endogenous IAPs to effectively block caspase activation and apoptosis in naïve cells. (A) Cytosolic lysates from naïve cells were incubated with serial dilutions of cytochrome c to identify a concentration of cytochrome c that was suboptimum for caspase activation in these extracts. (B) Naïve cytosolic extracts were incubated with a suboptimum dose of cytochrome c (100 nM) and 1 μM of either wild-type AVPI-Smac or mutant MVPI-Smac. Data shown are representative of multiple experiments. (C) Naïve PC12 cells were injected with serial dilutions of cytochrome c to identify a dose of cytochrome c that was suboptimum for inducing cell death. (D) Naïve cells were injected with a suboptimum dose of cytochrome c (100 μg/ml) and 1 mg/ml of either wild-type AVPI-Smac or mutant MVPI-Smac. Data shown are mean ± SEM of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172554&req=5

fig4: Reducing the apoptosome efficiency with addition of suboptimum cytochrome c permits endogenous IAPs to effectively block caspase activation and apoptosis in naïve cells. (A) Cytosolic lysates from naïve cells were incubated with serial dilutions of cytochrome c to identify a concentration of cytochrome c that was suboptimum for caspase activation in these extracts. (B) Naïve cytosolic extracts were incubated with a suboptimum dose of cytochrome c (100 nM) and 1 μM of either wild-type AVPI-Smac or mutant MVPI-Smac. Data shown are representative of multiple experiments. (C) Naïve PC12 cells were injected with serial dilutions of cytochrome c to identify a dose of cytochrome c that was suboptimum for inducing cell death. (D) Naïve cells were injected with a suboptimum dose of cytochrome c (100 μg/ml) and 1 mg/ml of either wild-type AVPI-Smac or mutant MVPI-Smac. Data shown are mean ± SEM of three independent experiments.

Mentions: To reduce apoptosome function in naïve cells, we simply decreased the amount of exogenous cytochrome c, thereby limiting the amount of apoptosome formation that could occur in these cells. Serial dilutions of cytochrome c were added to cytosolic lysate from naïve PC12 cells, causing a dose-dependent decrease in caspase activation (Fig. 4 A). To determine whether the reduced caspase activation was because of an increased ability of endogenous IAPs to inhibit caspase activation in these cells, we examined whether relieving IAP inhibition with Smac addition enhanced caspase activation seen with suboptimum cytochrome c. Indeed, addition of wild-type AVPI-Smac to lysates with suboptimum cytochrome c (100 nM) effectively increased the amount of caspase activation as compared with lysates with suboptimum cytochrome c alone (Fig. 4 B).


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)

Reducing the apoptosome efficiency with addition of suboptimum cytochrome c permits endogenous IAPs to effectively block caspase activation and apoptosis in naïve cells. (A) Cytosolic lysates from naïve cells were incubated with serial dilutions of cytochrome c to identify a concentration of cytochrome c that was suboptimum for caspase activation in these extracts. (B) Naïve cytosolic extracts were incubated with a suboptimum dose of cytochrome c (100 nM) and 1 μM of either wild-type AVPI-Smac or mutant MVPI-Smac. Data shown are representative of multiple experiments. (C) Naïve PC12 cells were injected with serial dilutions of cytochrome c to identify a dose of cytochrome c that was suboptimum for inducing cell death. (D) Naïve cells were injected with a suboptimum dose of cytochrome c (100 μg/ml) and 1 mg/ml of either wild-type AVPI-Smac or mutant MVPI-Smac. Data shown are mean ± SEM of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Reducing the apoptosome efficiency with addition of suboptimum cytochrome c permits endogenous IAPs to effectively block caspase activation and apoptosis in naïve cells. (A) Cytosolic lysates from naïve cells were incubated with serial dilutions of cytochrome c to identify a concentration of cytochrome c that was suboptimum for caspase activation in these extracts. (B) Naïve cytosolic extracts were incubated with a suboptimum dose of cytochrome c (100 nM) and 1 μM of either wild-type AVPI-Smac or mutant MVPI-Smac. Data shown are representative of multiple experiments. (C) Naïve PC12 cells were injected with serial dilutions of cytochrome c to identify a dose of cytochrome c that was suboptimum for inducing cell death. (D) Naïve cells were injected with a suboptimum dose of cytochrome c (100 μg/ml) and 1 mg/ml of either wild-type AVPI-Smac or mutant MVPI-Smac. Data shown are mean ± SEM of three independent experiments.
Mentions: To reduce apoptosome function in naïve cells, we simply decreased the amount of exogenous cytochrome c, thereby limiting the amount of apoptosome formation that could occur in these cells. Serial dilutions of cytochrome c were added to cytosolic lysate from naïve PC12 cells, causing a dose-dependent decrease in caspase activation (Fig. 4 A). To determine whether the reduced caspase activation was because of an increased ability of endogenous IAPs to inhibit caspase activation in these cells, we examined whether relieving IAP inhibition with Smac addition enhanced caspase activation seen with suboptimum cytochrome c. Indeed, addition of wild-type AVPI-Smac to lysates with suboptimum cytochrome c (100 nM) effectively increased the amount of caspase activation as compared with lysates with suboptimum cytochrome c alone (Fig. 4 B).

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