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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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Addition of exogenous cytochrome c induces caspase activation and apoptosis in naïve but not neuronally differentiated PC12 cells. (A) Naïve and neuronally differentiated PC12 cells were injected with 10–15 mg/ml cytochrome c or rhodamine dextran alone. Injected cells (arrows) were identified by the presence of rhodamine dextran coinjected with the cytochrome c. (B) Viability of naïve and neuronally differentiated cells at multiple times after the injections. Data shown are mean ± SEM of three independent experiments. (C) Cytosolic lysates from naïve and neuronally differentiated PC12 cells were assessed for caspase activation after the addition of 10 μM cytochrome c by measuring cleavage of the fluorogenic caspase substrate DEVD-afc. 10 μM yeast cytochrome c or 7 U granzyme B was added to lysates as a negative or positive control, respectively, for caspase activation. (D) Western analysis showing the status of caspase-9 (top) and caspase-3 (bottom) cleavage in cytosolic lysates from naïve and neuronally differentiated cells treated as described in C. Data shown are representative of at least three independent experiments.
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fig1: Addition of exogenous cytochrome c induces caspase activation and apoptosis in naïve but not neuronally differentiated PC12 cells. (A) Naïve and neuronally differentiated PC12 cells were injected with 10–15 mg/ml cytochrome c or rhodamine dextran alone. Injected cells (arrows) were identified by the presence of rhodamine dextran coinjected with the cytochrome c. (B) Viability of naïve and neuronally differentiated cells at multiple times after the injections. Data shown are mean ± SEM of three independent experiments. (C) Cytosolic lysates from naïve and neuronally differentiated PC12 cells were assessed for caspase activation after the addition of 10 μM cytochrome c by measuring cleavage of the fluorogenic caspase substrate DEVD-afc. 10 μM yeast cytochrome c or 7 U granzyme B was added to lysates as a negative or positive control, respectively, for caspase activation. (D) Western analysis showing the status of caspase-9 (top) and caspase-3 (bottom) cleavage in cytosolic lysates from naïve and neuronally differentiated cells treated as described in C. Data shown are representative of at least three independent experiments.

Mentions: PC12 cells can exist either as mitotic naïve cells, or can be differentiated with NGF treatment into postmitotic, neuronal-like cells (Greene and Tischler, 1976; Greene, 1978). Both naïve and neuronally differentiated PC12 cells undergo a Bcl-2 inhibitable, caspase-dependent apoptosis in response to a variety of apoptotic stimuli (Mesner et al., 1992; Batistatou et al., 1993; Pittman et al., 1993; Mills et al., 1995; Troy et al., 1996). To determine whether cellular differentiation of mitotic cells into postmitotic neurons induces a change in the postcytochrome c regulation of apoptosis, we examined whether cytosolic cytochrome c was sufficient to induce apoptosis in naïve or neuronally differentiated PC12 cells. Naïve PC12 cells were differentiated with NGF for 12 d, during which the cells become postmitotic and extend neurites (Fig. 1 A) (Greene and Tischler, 1976; Pittman et al., 1993).


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)

Addition of exogenous cytochrome c induces caspase activation and apoptosis in naïve but not neuronally differentiated PC12 cells. (A) Naïve and neuronally differentiated PC12 cells were injected with 10–15 mg/ml cytochrome c or rhodamine dextran alone. Injected cells (arrows) were identified by the presence of rhodamine dextran coinjected with the cytochrome c. (B) Viability of naïve and neuronally differentiated cells at multiple times after the injections. Data shown are mean ± SEM of three independent experiments. (C) Cytosolic lysates from naïve and neuronally differentiated PC12 cells were assessed for caspase activation after the addition of 10 μM cytochrome c by measuring cleavage of the fluorogenic caspase substrate DEVD-afc. 10 μM yeast cytochrome c or 7 U granzyme B was added to lysates as a negative or positive control, respectively, for caspase activation. (D) Western analysis showing the status of caspase-9 (top) and caspase-3 (bottom) cleavage in cytosolic lysates from naïve and neuronally differentiated cells treated as described in C. Data shown are representative of at least three independent experiments.
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Related In: Results  -  Collection

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

fig1: Addition of exogenous cytochrome c induces caspase activation and apoptosis in naïve but not neuronally differentiated PC12 cells. (A) Naïve and neuronally differentiated PC12 cells were injected with 10–15 mg/ml cytochrome c or rhodamine dextran alone. Injected cells (arrows) were identified by the presence of rhodamine dextran coinjected with the cytochrome c. (B) Viability of naïve and neuronally differentiated cells at multiple times after the injections. Data shown are mean ± SEM of three independent experiments. (C) Cytosolic lysates from naïve and neuronally differentiated PC12 cells were assessed for caspase activation after the addition of 10 μM cytochrome c by measuring cleavage of the fluorogenic caspase substrate DEVD-afc. 10 μM yeast cytochrome c or 7 U granzyme B was added to lysates as a negative or positive control, respectively, for caspase activation. (D) Western analysis showing the status of caspase-9 (top) and caspase-3 (bottom) cleavage in cytosolic lysates from naïve and neuronally differentiated cells treated as described in C. Data shown are representative of at least three independent experiments.
Mentions: PC12 cells can exist either as mitotic naïve cells, or can be differentiated with NGF treatment into postmitotic, neuronal-like cells (Greene and Tischler, 1976; Greene, 1978). Both naïve and neuronally differentiated PC12 cells undergo a Bcl-2 inhibitable, caspase-dependent apoptosis in response to a variety of apoptotic stimuli (Mesner et al., 1992; Batistatou et al., 1993; Pittman et al., 1993; Mills et al., 1995; Troy et al., 1996). To determine whether cellular differentiation of mitotic cells into postmitotic neurons induces a change in the postcytochrome c regulation of apoptosis, we examined whether cytosolic cytochrome c was sufficient to induce apoptosis in naïve or neuronally differentiated PC12 cells. Naïve PC12 cells were differentiated with NGF for 12 d, during which the cells become postmitotic and extend neurites (Fig. 1 A) (Greene and Tischler, 1976; Pittman et al., 1993).

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