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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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Reduction of Apaf-1 in primary fibroblasts allows strict IAP-mediated regulation of cytochrome c–mediated caspase activation. Cytosolic lysates were prepared from primary dermal fibroblasts from either wild-type (A) or Apaf-1 heterozygous (B) mice. The ability of bovine cytochrome c (10 μM) alone or bovine cytochrome c and Smac (1 μM) together to activate caspases was examined in these lysates. Yeast cytochrome c was added to these lysates as a negative control. Data shown are representative of at least three independent experiments.
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fig6: Reduction of Apaf-1 in primary fibroblasts allows strict IAP-mediated regulation of cytochrome c–mediated caspase activation. Cytosolic lysates were prepared from primary dermal fibroblasts from either wild-type (A) or Apaf-1 heterozygous (B) mice. The ability of bovine cytochrome c (10 μM) alone or bovine cytochrome c and Smac (1 μM) together to activate caspases was examined in these lysates. Yeast cytochrome c was added to these lysates as a negative control. Data shown are representative of at least three independent experiments.

Mentions: To test whether our model of coupling between Apaf-1 levels and IAP function can extend beyond the PC12 cell paradigm, we examined this in two different primary cell types. Our model predicts that reducing Apaf-1 levels in a primary cell would engage a strict regulation of cytochrome c–mediated apoptosis by endogenous IAPs where such a regulation is otherwise not detected. We examined this in cytosolic extracts of primary dermal fibroblasts isolated from wild-type and Apaf-1 heterozygous mice. Exogenous cytochrome c induced robust caspase activation in wild-type fibroblast lysates and addition of Smac did not enhance caspase activation, indicating that endogenous IAPs were ineffective in regulating caspase activation in wild-type fibroblasts (Fig. 6 A). In contrast, caspase activation with cytochrome c was significantly reduced in Apaf-1 heterozygous fibroblast lysates. Importantly, this reduction in cytochrome c–mediated caspase activation was not simply because of reduced Apaf-1 levels alone, but rather as a consequence of the resulting increased regulation by IAPs, as addition of Smac allowed greater activation of caspases in Apaf-1 heterozygous extracts (Fig. 6 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)

Reduction of Apaf-1 in primary fibroblasts allows strict IAP-mediated regulation of cytochrome c–mediated caspase activation. Cytosolic lysates were prepared from primary dermal fibroblasts from either wild-type (A) or Apaf-1 heterozygous (B) mice. The ability of bovine cytochrome c (10 μM) alone or bovine cytochrome c and Smac (1 μM) together to activate caspases was examined in these lysates. Yeast cytochrome c was added to these lysates as a negative control. Data shown are representative of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Reduction of Apaf-1 in primary fibroblasts allows strict IAP-mediated regulation of cytochrome c–mediated caspase activation. Cytosolic lysates were prepared from primary dermal fibroblasts from either wild-type (A) or Apaf-1 heterozygous (B) mice. The ability of bovine cytochrome c (10 μM) alone or bovine cytochrome c and Smac (1 μM) together to activate caspases was examined in these lysates. Yeast cytochrome c was added to these lysates as a negative control. Data shown are representative of at least three independent experiments.
Mentions: To test whether our model of coupling between Apaf-1 levels and IAP function can extend beyond the PC12 cell paradigm, we examined this in two different primary cell types. Our model predicts that reducing Apaf-1 levels in a primary cell would engage a strict regulation of cytochrome c–mediated apoptosis by endogenous IAPs where such a regulation is otherwise not detected. We examined this in cytosolic extracts of primary dermal fibroblasts isolated from wild-type and Apaf-1 heterozygous mice. Exogenous cytochrome c induced robust caspase activation in wild-type fibroblast lysates and addition of Smac did not enhance caspase activation, indicating that endogenous IAPs were ineffective in regulating caspase activation in wild-type fibroblasts (Fig. 6 A). In contrast, caspase activation with cytochrome c was significantly reduced in Apaf-1 heterozygous fibroblast lysates. Importantly, this reduction in cytochrome c–mediated caspase activation was not simply because of reduced Apaf-1 levels alone, but rather as a consequence of the resulting increased regulation by IAPs, as addition of Smac allowed greater activation of caspases in Apaf-1 heterozygous extracts (Fig. 6 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