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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-mediated induction of Bcl-2 family members 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 normalized 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, (2 μg/ml) Tm for indicated time points, and induction of Bim, and CHOP was determined by western blot analysis. β-actin was used to determine equal loading of samples.
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fig5: ER stress-mediated induction of Bcl-2 family members 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 normalized 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, (2 μg/ml) Tm for indicated time points, and induction of Bim, and CHOP was determined by western blot analysis. β-actin was used to determine equal loading of samples.

Mentions: The mitochondrial apoptotic signalling pathway involves activation of the proapoptotic Bcl-2 family members Bax and Bak, that induce permeabilization of the mitochondrial outer membrane and release of cytochrome c [18]. To investigate the involvement of Bcl-2 family proteins in ER stress-induced cell death, we determined the effect of ER stress on the expression levels of Bcl-2 family members in H9c2 cells (Figure 5(a)). Our studies demonstrated that while Noxa, Mcl1, and Bax were upregulated by all three ER stress-inducing agents (Tg, Tm and BFA), Bim showed the greatest fold changes in response to any type of ER stress. The upregulation of Bim protein upon ER stress was confirmed by Western blotting (Figure 5(b)). We observed that Tg was most effective in inducing Bim mRNA levels; however, Tm was more potent in inducing Bim protein levels. This could be due to the posttranslational modifications regulating BIM protein stability upon exposure to ER stress [22]. In order to test the function of Bcl-2 on the mitochondria, we used the cell permeable BH4-Tat peptide. The BH4 domain of antiapoptotic Bcl-2 family members accumulates on the mitochondria and inhibits cell death [32]. Pretreatment of cells with BH4-Tat protected the mitochondria against the effect of Tg, delaying membrane depolarisation by at least 12 hours (Figure 6(a)). Next we generated a Bcl-2 overexpressing H9c2 clone and examined the protective potential of Bcl-2 in these cells. Bcl-2 overexpression was able to prevent loss of ΔΨm upon Tg treatment, for at least 48 hours (Figure 6(a)). Furthermore, Bcl-2 overexpression efficiently protected cells against Tm induced cell death (Figure 6(d)), whereas pretreatment of cells with BH4-Tat was not able to inhibit ER stress-induced apoptosis (Figure 6(c)).


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-mediated induction of Bcl-2 family members 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 normalized 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, (2 μg/ml) Tm for indicated time points, and induction of Bim, and CHOP was determined by western blot analysis. β-actin was used to determine equal loading of samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2821636&req=5

fig5: ER stress-mediated induction of Bcl-2 family members 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 normalized 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, (2 μg/ml) Tm for indicated time points, and induction of Bim, and CHOP was determined by western blot analysis. β-actin was used to determine equal loading of samples.
Mentions: The mitochondrial apoptotic signalling pathway involves activation of the proapoptotic Bcl-2 family members Bax and Bak, that induce permeabilization of the mitochondrial outer membrane and release of cytochrome c [18]. To investigate the involvement of Bcl-2 family proteins in ER stress-induced cell death, we determined the effect of ER stress on the expression levels of Bcl-2 family members in H9c2 cells (Figure 5(a)). Our studies demonstrated that while Noxa, Mcl1, and Bax were upregulated by all three ER stress-inducing agents (Tg, Tm and BFA), Bim showed the greatest fold changes in response to any type of ER stress. The upregulation of Bim protein upon ER stress was confirmed by Western blotting (Figure 5(b)). We observed that Tg was most effective in inducing Bim mRNA levels; however, Tm was more potent in inducing Bim protein levels. This could be due to the posttranslational modifications regulating BIM protein stability upon exposure to ER stress [22]. In order to test the function of Bcl-2 on the mitochondria, we used the cell permeable BH4-Tat peptide. The BH4 domain of antiapoptotic Bcl-2 family members accumulates on the mitochondria and inhibits cell death [32]. Pretreatment of cells with BH4-Tat protected the mitochondria against the effect of Tg, delaying membrane depolarisation by at least 12 hours (Figure 6(a)). Next we generated a Bcl-2 overexpressing H9c2 clone and examined the protective potential of Bcl-2 in these cells. Bcl-2 overexpression was able to prevent loss of ΔΨm upon Tg treatment, for at least 48 hours (Figure 6(a)). Furthermore, Bcl-2 overexpression efficiently protected cells against Tm induced cell death (Figure 6(d)), whereas pretreatment of cells with BH4-Tat was not able to inhibit ER stress-induced apoptosis (Figure 6(c)).

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