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Mechanisms of ER Stress-Mediated Mitochondrial Membrane Permeabilization.

Gupta S, Cuffe L, Szegezdi E, Logue SE, Neary C, Healy S, Samali A - Int J Cell Biol (2010)

Bottom Line: Furthermore, pretreatment of cells with caspase inhibitors (Boc-D.fmk and DEVD.fmk) attenuated ER stress-induced loss of DeltaPsim.Bcl-2 overexpression or pretreatment of cells with the cell permeable BH4 domain (BH4-Tat) or the mitochondrial permeability transition pore inhibitors, bongkrekic acid or cyclosporine A, attenuated the ER stress-induced loss of DeltaPsim.These data suggest a role for caspase-9 and -2, Bcl-2 family members and the mitochondrial permeability transition pore in loss of mitochondrial membrane potential during ER stress-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Apoptosis Research Centre, School of Natural Sciences, National University of Ireland, Galway, Ireland.

ABSTRACT
During apoptosis, the process of mitochondrial outer membrane permeabilization (MOMP) represents a point-of-no-return as it commits the cell to death. Here we have assessed the role of caspases, Bcl-2 family members and the mitochondrial permeability transition pore on ER stress-induced MOMP and subsequent cell death. Induction of ER stress leads to upregulation of several genes such as Grp78, Edem1, Erp72, Atf4, Wars, Herp, p58ipk, and ERdj4 and leads to caspase activation, release of mitochondrial intermembrane proteins and dissipation of mitochondrial transmembrane potential (DeltaPsim). Mouse embryonic fibroblasts (MEFs) from caspase-9, -2 and, -3 knock-out mice were resistant to ER stress-induced apoptosis which correlated with decreased processing of pro-caspase-3 and -9. Furthermore, pretreatment of cells with caspase inhibitors (Boc-D.fmk and DEVD.fmk) attenuated ER stress-induced loss of DeltaPsim. However, only deficiency of caspase-9 and -2 could prevent ER stress-mediated loss of DeltaPsim. Bcl-2 overexpression or pretreatment of cells with the cell permeable BH4 domain (BH4-Tat) or the mitochondrial permeability transition pore inhibitors, bongkrekic acid or cyclosporine A, attenuated the ER stress-induced loss of DeltaPsim. These data suggest a role for caspase-9 and -2, Bcl-2 family members and the mitochondrial permeability transition pore in loss of mitochondrial membrane potential during ER stress-induced apoptosis.

No MeSH data available.


Loss of mitochondrial membrane potential and release of cytochrome c during ER stress-induced apoptosis. (a) H9c2 cells were treated with (2 μM) Tg for the indicated times. Following treatment, cells were incubated with (100 nM) TMRE. Mitochondrial membrane potential was monitored by measuring the fluorescence intensity at 582 nm (FL2). As a positive control for depletion of membrane potential, cells were treated with (10 μM) CCCP for 45 minutes. The data is a representative of at least three independent experiments.  (b) Western blot analysis of cytochrome c and Smac in cytosolic fractions. The data is a representative of at least three independent experiments.
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fig2: Loss of mitochondrial membrane potential and release of cytochrome c during ER stress-induced apoptosis. (a) H9c2 cells were treated with (2 μM) Tg for the indicated times. Following treatment, cells were incubated with (100 nM) TMRE. Mitochondrial membrane potential was monitored by measuring the fluorescence intensity at 582 nm (FL2). As a positive control for depletion of membrane potential, cells were treated with (10 μM) CCCP for 45 minutes. The data is a representative of at least three independent experiments. (b) Western blot analysis of cytochrome c and Smac in cytosolic fractions. The data is a representative of at least three independent experiments.

Mentions: Several reports have indicated a possible role for mitochondria in ER stress-induced apoptosis [10, 27]. To determine the exact contribution of the mitochondria to ER stress-induced apoptosis, alterations of ΔΨm and release of mitochondrial intermembrane space proteins into the cytosol were analyzed. Alterations in ΔΨm were studied using tetramethyl rhodamine ethyl ester (TMRE). The loss of ΔΨm was detectable by flow cytometry starting after 24 hours of induction of ER stress and increasing over time (Figure 2(a)). Consistent with a drop in ΔΨm, Western blot analysis demonstrated increased cytosolic levels of cytochrome c and Smac, when compared to untreated controls, at 36 and 48 hours post Tg treatment (Figure 2(b)). The slight differences observed in the kinetics of release from mitochondria between cytochrome c and SMAC may be due the differences in the affinity of cytochrome c and SMAC antibodies used for western blotting. These results show that ER stress induces MOMP and the release of proapoptotic proteins from the intermembrane space into the cytosol. These observations suggest that the loss of ΔΨm and release of mitochondrial intermembrane space proteins into the cytosol are coupled and a component of ER stress-induced apoptosis.


Mechanisms of ER Stress-Mediated Mitochondrial Membrane Permeabilization.

Gupta S, Cuffe L, Szegezdi E, Logue SE, Neary C, Healy S, Samali A - Int J Cell Biol (2010)

Loss of mitochondrial membrane potential and release of cytochrome c during ER stress-induced apoptosis. (a) H9c2 cells were treated with (2 μM) Tg for the indicated times. Following treatment, cells were incubated with (100 nM) TMRE. Mitochondrial membrane potential was monitored by measuring the fluorescence intensity at 582 nm (FL2). As a positive control for depletion of membrane potential, cells were treated with (10 μM) CCCP for 45 minutes. The data is a representative of at least three independent experiments.  (b) Western blot analysis of cytochrome c and Smac in cytosolic fractions. The data is a representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Loss of mitochondrial membrane potential and release of cytochrome c during ER stress-induced apoptosis. (a) H9c2 cells were treated with (2 μM) Tg for the indicated times. Following treatment, cells were incubated with (100 nM) TMRE. Mitochondrial membrane potential was monitored by measuring the fluorescence intensity at 582 nm (FL2). As a positive control for depletion of membrane potential, cells were treated with (10 μM) CCCP for 45 minutes. The data is a representative of at least three independent experiments. (b) Western blot analysis of cytochrome c and Smac in cytosolic fractions. The data is a representative of at least three independent experiments.
Mentions: Several reports have indicated a possible role for mitochondria in ER stress-induced apoptosis [10, 27]. To determine the exact contribution of the mitochondria to ER stress-induced apoptosis, alterations of ΔΨm and release of mitochondrial intermembrane space proteins into the cytosol were analyzed. Alterations in ΔΨm were studied using tetramethyl rhodamine ethyl ester (TMRE). The loss of ΔΨm was detectable by flow cytometry starting after 24 hours of induction of ER stress and increasing over time (Figure 2(a)). Consistent with a drop in ΔΨm, Western blot analysis demonstrated increased cytosolic levels of cytochrome c and Smac, when compared to untreated controls, at 36 and 48 hours post Tg treatment (Figure 2(b)). The slight differences observed in the kinetics of release from mitochondria between cytochrome c and SMAC may be due the differences in the affinity of cytochrome c and SMAC antibodies used for western blotting. These results show that ER stress induces MOMP and the release of proapoptotic proteins from the intermembrane space into the cytosol. These observations suggest that the loss of ΔΨm and release of mitochondrial intermembrane space proteins into the cytosol are coupled and a component of ER stress-induced apoptosis.

Bottom Line: Furthermore, pretreatment of cells with caspase inhibitors (Boc-D.fmk and DEVD.fmk) attenuated ER stress-induced loss of DeltaPsim.Bcl-2 overexpression or pretreatment of cells with the cell permeable BH4 domain (BH4-Tat) or the mitochondrial permeability transition pore inhibitors, bongkrekic acid or cyclosporine A, attenuated the ER stress-induced loss of DeltaPsim.These data suggest a role for caspase-9 and -2, Bcl-2 family members and the mitochondrial permeability transition pore in loss of mitochondrial membrane potential during ER stress-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Apoptosis Research Centre, School of Natural Sciences, National University of Ireland, Galway, Ireland.

ABSTRACT
During apoptosis, the process of mitochondrial outer membrane permeabilization (MOMP) represents a point-of-no-return as it commits the cell to death. Here we have assessed the role of caspases, Bcl-2 family members and the mitochondrial permeability transition pore on ER stress-induced MOMP and subsequent cell death. Induction of ER stress leads to upregulation of several genes such as Grp78, Edem1, Erp72, Atf4, Wars, Herp, p58ipk, and ERdj4 and leads to caspase activation, release of mitochondrial intermembrane proteins and dissipation of mitochondrial transmembrane potential (DeltaPsim). Mouse embryonic fibroblasts (MEFs) from caspase-9, -2 and, -3 knock-out mice were resistant to ER stress-induced apoptosis which correlated with decreased processing of pro-caspase-3 and -9. Furthermore, pretreatment of cells with caspase inhibitors (Boc-D.fmk and DEVD.fmk) attenuated ER stress-induced loss of DeltaPsim. However, only deficiency of caspase-9 and -2 could prevent ER stress-mediated loss of DeltaPsim. Bcl-2 overexpression or pretreatment of cells with the cell permeable BH4 domain (BH4-Tat) or the mitochondrial permeability transition pore inhibitors, bongkrekic acid or cyclosporine A, attenuated the ER stress-induced loss of DeltaPsim. These data suggest a role for caspase-9 and -2, Bcl-2 family members and the mitochondrial permeability transition pore in loss of mitochondrial membrane potential during ER stress-induced apoptosis.

No MeSH data available.