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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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Exogenous addition of Smac that can relieve IAP inhibition permits cytochrome c to activate caspases and induce death in neuronally differentiated PC12 cells. Neuronally differentiated (A and B) or naïve (C and D) PC12 cells were treated with either cytochrome c alone, cytochrome c and wild-type AVPI-Smac, or cytochrome c and mutant MVPI-Smac. These proteins were either added to cytosolic lysates prepared from naïve or neuronally differentiated cells (Cyt c, 10 μM; Smac, 1 μM) and assessed for their ability to activate caspases by the DEVD-afc cleavage assay (A and C) or injected into intact naïve or neuronally differentiated cells (Cyt c, 10 mg/ml; Smac, 1 mg/ml) and assessed for their ability to induce cell death (B and D). Data shown are mean ± SEM (B and D) or representative (A and C) of at least three independent experiments.
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fig2: Exogenous addition of Smac that can relieve IAP inhibition permits cytochrome c to activate caspases and induce death in neuronally differentiated PC12 cells. Neuronally differentiated (A and B) or naïve (C and D) PC12 cells were treated with either cytochrome c alone, cytochrome c and wild-type AVPI-Smac, or cytochrome c and mutant MVPI-Smac. These proteins were either added to cytosolic lysates prepared from naïve or neuronally differentiated cells (Cyt c, 10 μM; Smac, 1 μM) and assessed for their ability to activate caspases by the DEVD-afc cleavage assay (A and C) or injected into intact naïve or neuronally differentiated cells (Cyt c, 10 mg/ml; Smac, 1 mg/ml) and assessed for their ability to induce cell death (B and D). Data shown are mean ± SEM (B and D) or representative (A and C) of at least three independent experiments.

Mentions: In primary sympathetic neurons, the resistance to cytochrome c–induced apoptosis is mediated by endogenous IAPs (Deshmukh et al., 2002; Potts et al., 2003). To determine whether a similar IAP-mediated regulation was promoting resistance to cytochrome c in neuronally differentiated PC12 cells, we examined whether addition of Smac, an IAP inhibitor, could relieve the inhibition and permit cytochrome c to activate caspases in these cells. Addition of wild-type mature Smac (AVPI-Smac) and cytochrome c together resulted in increased caspase activation in cytosolic extracts from neuronally differentiated cells that are otherwise resistant to addition of cytochrome c or AVPI-Smac alone (Fig. 2 A; unpublished data). The concentration of Smac used in these experiments was maximal, as a 10-fold increase in Smac did not result in any further increase in caspase activation in cell extracts. Importantly, unlike wild-type AVPI-Smac, mutant MVPI-Smac, which is unable to bind to IAPs and inhibit their function (Chai et al., 2000), did not enhance cytochrome c–mediated caspase activation in these cell extracts (Fig. 2 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)

Exogenous addition of Smac that can relieve IAP inhibition permits cytochrome c to activate caspases and induce death in neuronally differentiated PC12 cells. Neuronally differentiated (A and B) or naïve (C and D) PC12 cells were treated with either cytochrome c alone, cytochrome c and wild-type AVPI-Smac, or cytochrome c and mutant MVPI-Smac. These proteins were either added to cytosolic lysates prepared from naïve or neuronally differentiated cells (Cyt c, 10 μM; Smac, 1 μM) and assessed for their ability to activate caspases by the DEVD-afc cleavage assay (A and C) or injected into intact naïve or neuronally differentiated cells (Cyt c, 10 mg/ml; Smac, 1 mg/ml) and assessed for their ability to induce cell death (B and D). Data shown are mean ± SEM (B and D) or representative (A and C) of at least three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172554&req=5

fig2: Exogenous addition of Smac that can relieve IAP inhibition permits cytochrome c to activate caspases and induce death in neuronally differentiated PC12 cells. Neuronally differentiated (A and B) or naïve (C and D) PC12 cells were treated with either cytochrome c alone, cytochrome c and wild-type AVPI-Smac, or cytochrome c and mutant MVPI-Smac. These proteins were either added to cytosolic lysates prepared from naïve or neuronally differentiated cells (Cyt c, 10 μM; Smac, 1 μM) and assessed for their ability to activate caspases by the DEVD-afc cleavage assay (A and C) or injected into intact naïve or neuronally differentiated cells (Cyt c, 10 mg/ml; Smac, 1 mg/ml) and assessed for their ability to induce cell death (B and D). Data shown are mean ± SEM (B and D) or representative (A and C) of at least three independent experiments.
Mentions: In primary sympathetic neurons, the resistance to cytochrome c–induced apoptosis is mediated by endogenous IAPs (Deshmukh et al., 2002; Potts et al., 2003). To determine whether a similar IAP-mediated regulation was promoting resistance to cytochrome c in neuronally differentiated PC12 cells, we examined whether addition of Smac, an IAP inhibitor, could relieve the inhibition and permit cytochrome c to activate caspases in these cells. Addition of wild-type mature Smac (AVPI-Smac) and cytochrome c together resulted in increased caspase activation in cytosolic extracts from neuronally differentiated cells that are otherwise resistant to addition of cytochrome c or AVPI-Smac alone (Fig. 2 A; unpublished data). The concentration of Smac used in these experiments was maximal, as a 10-fold increase in Smac did not result in any further increase in caspase activation in cell extracts. Importantly, unlike wild-type AVPI-Smac, mutant MVPI-Smac, which is unable to bind to IAPs and inhibit their function (Chai et al., 2000), did not enhance cytochrome c–mediated caspase activation in these cell extracts (Fig. 2 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