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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.


Related in: MedlinePlus

Resistance to ER stress-induced death and loss of mitochondrial membrane potential in absence of caspases. (a)–(c) Left panel, Indicated MEFs were treated with (2 μM) Tg for 24 hours. Cells were stained with haematoxylin-eosin-stain and visualised using an Olympus IX71 microscope at 40×. Images are representative of 2 independent experiments. Right panel, indicated MEFs were treated with (2 μM) Tg for the indicated times. Increase in cell death was measured by annexin V staining. The data is representative of at least 2 independent experiments. (d) H9c2 cells were treated with (2 μM) Tg alone or pretreated for 30 minutes with Boc-D.fmk (20 μM) and DEVD.fmk (20 μM) prior to treatment with Tg, for the indicated time periods. 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. (e)–(f) Indicated MEFs were treated with (2 μM) Tg for 24 hours. Following treatment cells were incubated with (100 nM) TMRE. Mitochondrial membrane potential was monitored by measuring the fluorescence intensity at 582 nm (FL2).  The data is a representative of at least three independent experiments.
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fig3: Resistance to ER stress-induced death and loss of mitochondrial membrane potential in absence of caspases. (a)–(c) Left panel, Indicated MEFs were treated with (2 μM) Tg for 24 hours. Cells were stained with haematoxylin-eosin-stain and visualised using an Olympus IX71 microscope at 40×. Images are representative of 2 independent experiments. Right panel, indicated MEFs were treated with (2 μM) Tg for the indicated times. Increase in cell death was measured by annexin V staining. The data is representative of at least 2 independent experiments. (d) H9c2 cells were treated with (2 μM) Tg alone or pretreated for 30 minutes with Boc-D.fmk (20 μM) and DEVD.fmk (20 μM) prior to treatment with Tg, for the indicated time periods. 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. (e)–(f) Indicated MEFs were treated with (2 μM) Tg for 24 hours. Following treatment cells were incubated with (100 nM) TMRE. Mitochondrial membrane potential was monitored by measuring the fluorescence intensity at 582 nm (FL2). The data is a representative of at least three independent experiments.

Mentions: In Tg-treated H9c2 cells, we detected caspase-3 activation starting as early as 12–18 hours, which preceded detectable changes in the mitochondria. This suggested a possible involvement of caspases in inducing ΔΨm depletion. Caspase-3 and -9 are important in both the intrinsic and extrinsic pathways of apoptosis. To determine the role of these caspases in ER stress-induced cell death, we have used mouse embryonic fibroblasts (MEFs) deficient in caspase-3, -2, and -9. Fibroblasts from wild-type and homozygous knock-out embryos were treated with 2 μM Tg for 24 hours, and MEFs were assayed for viability using annexin V staining. As shown in Figures 3(a)–3(c), caspase-9, -2, and -3 knock-out MEFs were protected against apoptosis induced by Tg. Next we tested the effect of broad range caspase inhibitors on the ER stress-mediated drop in ∆Ψm. H9c2 cells were treated with Tg in the presence or absence of the caspase inhibitors Boc-D.fmk and DEVD.fmk (Figure 3(d)). Inhibition of caspases reduced Tg-induced loss of ΔΨm up to 36 hours, but loses effectiveness at 48 hours. To further confirm the role of caspases in ER stress-mediated ΔΨm loss, we determined mitochondrial membrane potential in caspase-9, -2, and -3 deficient and wild-type MEFs upon exposure to ER stress. We observed that caspase-9 and caspase-2 deficient MEFs showed resistance to ER stress-mediated loss of ΔΨm as compared to wild-type MEFs (Figure 3(e)). In contrast, caspase-3 deficient MEFs showed loss in ΔΨm comparable to wild-type MEFs (Figure 3(f)). These results suggest that caspase-2 and -9 but not caspase-3 play a role in the ΔΨm loss during ER stress-induced apoptosis. The apparent differences in ER stress-mediated loss of ΔΨm upon DEVD.fmk pretreatment (Figure 3(d)) and in caspase-3 deficient MEFs (Figure 3(f)) may be due to inhibition of both caspase-3 and -7 by DEVD.fmk. This is in agreement with a previous study that showed that early apoptoptic events (e.g., Bax translocation and cytochrome c release) following mitochondria-mediated apoptosis triggered by UV irradiation were compromised by a double knock-out of caspase-3 and -7 in MEFs, but not in caspase-3 knock-out cells [30].


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)

Resistance to ER stress-induced death and loss of mitochondrial membrane potential in absence of caspases. (a)–(c) Left panel, Indicated MEFs were treated with (2 μM) Tg for 24 hours. Cells were stained with haematoxylin-eosin-stain and visualised using an Olympus IX71 microscope at 40×. Images are representative of 2 independent experiments. Right panel, indicated MEFs were treated with (2 μM) Tg for the indicated times. Increase in cell death was measured by annexin V staining. The data is representative of at least 2 independent experiments. (d) H9c2 cells were treated with (2 μM) Tg alone or pretreated for 30 minutes with Boc-D.fmk (20 μM) and DEVD.fmk (20 μM) prior to treatment with Tg, for the indicated time periods. 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. (e)–(f) Indicated MEFs were treated with (2 μM) Tg for 24 hours. Following treatment cells were incubated with (100 nM) TMRE. Mitochondrial membrane potential was monitored by measuring the fluorescence intensity at 582 nm (FL2).  The data is a representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Resistance to ER stress-induced death and loss of mitochondrial membrane potential in absence of caspases. (a)–(c) Left panel, Indicated MEFs were treated with (2 μM) Tg for 24 hours. Cells were stained with haematoxylin-eosin-stain and visualised using an Olympus IX71 microscope at 40×. Images are representative of 2 independent experiments. Right panel, indicated MEFs were treated with (2 μM) Tg for the indicated times. Increase in cell death was measured by annexin V staining. The data is representative of at least 2 independent experiments. (d) H9c2 cells were treated with (2 μM) Tg alone or pretreated for 30 minutes with Boc-D.fmk (20 μM) and DEVD.fmk (20 μM) prior to treatment with Tg, for the indicated time periods. 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. (e)–(f) Indicated MEFs were treated with (2 μM) Tg for 24 hours. Following treatment cells were incubated with (100 nM) TMRE. Mitochondrial membrane potential was monitored by measuring the fluorescence intensity at 582 nm (FL2). The data is a representative of at least three independent experiments.
Mentions: In Tg-treated H9c2 cells, we detected caspase-3 activation starting as early as 12–18 hours, which preceded detectable changes in the mitochondria. This suggested a possible involvement of caspases in inducing ΔΨm depletion. Caspase-3 and -9 are important in both the intrinsic and extrinsic pathways of apoptosis. To determine the role of these caspases in ER stress-induced cell death, we have used mouse embryonic fibroblasts (MEFs) deficient in caspase-3, -2, and -9. Fibroblasts from wild-type and homozygous knock-out embryos were treated with 2 μM Tg for 24 hours, and MEFs were assayed for viability using annexin V staining. As shown in Figures 3(a)–3(c), caspase-9, -2, and -3 knock-out MEFs were protected against apoptosis induced by Tg. Next we tested the effect of broad range caspase inhibitors on the ER stress-mediated drop in ∆Ψm. H9c2 cells were treated with Tg in the presence or absence of the caspase inhibitors Boc-D.fmk and DEVD.fmk (Figure 3(d)). Inhibition of caspases reduced Tg-induced loss of ΔΨm up to 36 hours, but loses effectiveness at 48 hours. To further confirm the role of caspases in ER stress-mediated ΔΨm loss, we determined mitochondrial membrane potential in caspase-9, -2, and -3 deficient and wild-type MEFs upon exposure to ER stress. We observed that caspase-9 and caspase-2 deficient MEFs showed resistance to ER stress-mediated loss of ΔΨm as compared to wild-type MEFs (Figure 3(e)). In contrast, caspase-3 deficient MEFs showed loss in ΔΨm comparable to wild-type MEFs (Figure 3(f)). These results suggest that caspase-2 and -9 but not caspase-3 play a role in the ΔΨm loss during ER stress-induced apoptosis. The apparent differences in ER stress-mediated loss of ΔΨm upon DEVD.fmk pretreatment (Figure 3(d)) and in caspase-3 deficient MEFs (Figure 3(f)) may be due to inhibition of both caspase-3 and -7 by DEVD.fmk. This is in agreement with a previous study that showed that early apoptoptic events (e.g., Bax translocation and cytochrome c release) following mitochondria-mediated apoptosis triggered by UV irradiation were compromised by a double knock-out of caspase-3 and -7 in MEFs, but not in caspase-3 knock-out cells [30].

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.


Related in: MedlinePlus