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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

ER stress-induced apoptosis in H9c2 cells. (a) H9c2 cells were left untreated or treated with (2 μM) Tg, (2 μg/ml) Tm or (2 μg/ml) BFA for 24 hours. The change in expression levels of ER stress markers was measured by real-time RT-PCR normalizing against GAPDH expression and plotting expression levels relative to the control. Error bars represent mean ± SD from an experiment performed in duplicate and reproduced twice. (b) H9c2 cells were left untreated or treated with (2 μM) Tg for the indicated times. The induction of ER stress markers, Grp78, Grp94 and CHOP was determined by Western blot analysis. β-actin was used to determine equal loading of samples. (c) H9c2 cells were left untreated or treated with (2 μM) Tg for the indicated times. After 48 hours of Tg-treatment, cells were stained with haematoxylin-eosin-stain and photographed at 200 × magnification. (d) H9c2 cells were left untreated or treated with (2 μM) Tg for the indicated times. The processing of procaspase-3 and cleavage of PKCδ were determined by Western blot analysis. The caspase activity was determined using DEVD-AMC. The figure is a representative of three independent experiments.
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fig1: ER stress-induced apoptosis in H9c2 cells. (a) H9c2 cells were left untreated or treated with (2 μM) Tg, (2 μg/ml) Tm or (2 μg/ml) BFA for 24 hours. The change in expression levels of ER stress markers was measured by real-time RT-PCR normalizing against GAPDH expression and plotting expression levels relative to the control. Error bars represent mean ± SD from an experiment performed in duplicate and reproduced twice. (b) H9c2 cells were left untreated or treated with (2 μM) Tg for the indicated times. The induction of ER stress markers, Grp78, Grp94 and CHOP was determined by Western blot analysis. β-actin was used to determine equal loading of samples. (c) H9c2 cells were left untreated or treated with (2 μM) Tg for the indicated times. After 48 hours of Tg-treatment, cells were stained with haematoxylin-eosin-stain and photographed at 200 × magnification. (d) H9c2 cells were left untreated or treated with (2 μM) Tg for the indicated times. The processing of procaspase-3 and cleavage of PKCδ were determined by Western blot analysis. The caspase activity was determined using DEVD-AMC. The figure is a representative of three independent experiments.

Mentions: To induce ER stress, H9c2 cells, a neonatal rat cardiomyocyte-derived cell line, were treated with three different ER stress-inducing agents: thapsigargin (Tg) an inhibitor of the Sacroplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump, tunicamycin (Tm) an inhibitor of N-linked glycosylation, and brefeldin A (BFA) an inhibitor of protein transfer from the ER to the Golgi. Treatment of H9c2 cells with any of these ER stress inducing agents caused an increase in the mRNA levels of many genes associated with the ER stress response (Figure 1(a)). We also examined protein levels by Western blot analysis for a subset of these genes and found them to reflect the changes observed in mRNA expression with Grp78, Grp90, and the proapoptotic transcription factor CHOP/GADD153 being significantly upregulated after treating cells with Tg (Figure 1(b)). Conditions of prolonged (48 hours) ER stress induced morphological changes associated with apoptosis, including cellular shrinkage, nuclear condensation, and membrane blebbing (Figure 1(c)). ER stress-induced apoptosis was associated with activation of caspases as detected by the processing of caspase-3, an increase in DEVDase activity, and the cleavage of protein kinase C delta (PKCδ), a cellular substrate of caspases, which was detectable as early as 24 hours post Tg treatment (Figure 1(d)). Collectively, these data demonstrate that ER stress induces apoptotic cell death in H9c2 cardiomyocytes.


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)

ER stress-induced apoptosis in H9c2 cells. (a) H9c2 cells were left untreated or treated with (2 μM) Tg, (2 μg/ml) Tm or (2 μg/ml) BFA for 24 hours. The change in expression levels of ER stress markers was measured by real-time RT-PCR normalizing against GAPDH expression and plotting expression levels relative to the control. Error bars represent mean ± SD from an experiment performed in duplicate and reproduced twice. (b) H9c2 cells were left untreated or treated with (2 μM) Tg for the indicated times. The induction of ER stress markers, Grp78, Grp94 and CHOP was determined by Western blot analysis. β-actin was used to determine equal loading of samples. (c) H9c2 cells were left untreated or treated with (2 μM) Tg for the indicated times. After 48 hours of Tg-treatment, cells were stained with haematoxylin-eosin-stain and photographed at 200 × magnification. (d) H9c2 cells were left untreated or treated with (2 μM) Tg for the indicated times. The processing of procaspase-3 and cleavage of PKCδ were determined by Western blot analysis. The caspase activity was determined using DEVD-AMC. The figure is a representative of three independent experiments.
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Related In: Results  -  Collection

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fig1: ER stress-induced apoptosis in H9c2 cells. (a) H9c2 cells were left untreated or treated with (2 μM) Tg, (2 μg/ml) Tm or (2 μg/ml) BFA for 24 hours. The change in expression levels of ER stress markers was measured by real-time RT-PCR normalizing against GAPDH expression and plotting expression levels relative to the control. Error bars represent mean ± SD from an experiment performed in duplicate and reproduced twice. (b) H9c2 cells were left untreated or treated with (2 μM) Tg for the indicated times. The induction of ER stress markers, Grp78, Grp94 and CHOP was determined by Western blot analysis. β-actin was used to determine equal loading of samples. (c) H9c2 cells were left untreated or treated with (2 μM) Tg for the indicated times. After 48 hours of Tg-treatment, cells were stained with haematoxylin-eosin-stain and photographed at 200 × magnification. (d) H9c2 cells were left untreated or treated with (2 μM) Tg for the indicated times. The processing of procaspase-3 and cleavage of PKCδ were determined by Western blot analysis. The caspase activity was determined using DEVD-AMC. The figure is a representative of three independent experiments.
Mentions: To induce ER stress, H9c2 cells, a neonatal rat cardiomyocyte-derived cell line, were treated with three different ER stress-inducing agents: thapsigargin (Tg) an inhibitor of the Sacroplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump, tunicamycin (Tm) an inhibitor of N-linked glycosylation, and brefeldin A (BFA) an inhibitor of protein transfer from the ER to the Golgi. Treatment of H9c2 cells with any of these ER stress inducing agents caused an increase in the mRNA levels of many genes associated with the ER stress response (Figure 1(a)). We also examined protein levels by Western blot analysis for a subset of these genes and found them to reflect the changes observed in mRNA expression with Grp78, Grp90, and the proapoptotic transcription factor CHOP/GADD153 being significantly upregulated after treating cells with Tg (Figure 1(b)). Conditions of prolonged (48 hours) ER stress induced morphological changes associated with apoptosis, including cellular shrinkage, nuclear condensation, and membrane blebbing (Figure 1(c)). ER stress-induced apoptosis was associated with activation of caspases as detected by the processing of caspase-3, an increase in DEVDase activity, and the cleavage of protein kinase C delta (PKCδ), a cellular substrate of caspases, which was detectable as early as 24 hours post Tg treatment (Figure 1(d)). Collectively, these data demonstrate that ER stress induces apoptotic cell death in H9c2 cardiomyocytes.

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