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Necroptosis Interfaces with MOMP and the MPTP in Mediating Cell Death.

Karch J, Kanisicak O, Brody MJ, Sargent MA, Michael DM, Molkentin JD - PLoS ONE (2015)

Bottom Line: Mechanistically, we observe mixed lineage kinase domain-like (MLKL) protein and cofilin-1 translocation to the mitochondria following necroptosis induction, while expression of the mitochondrial matrix isoform of the antiapoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), is significantly reduced.Some of these effects are lost with necroptosis inhibition in Bax/Bak1 double , Ppif-/-, or Ripk3-/- fibroblasts.Hence, downstream mechanisms of cell death induced by necroptotic stimuli utilize both Bax/Bak to generate apoptotic pores in the outer mitochondrial membrane as well as MPTP opening in association with known mitochondrial death modifying proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, United States of America.

ABSTRACT
During apoptosis the pro-death Bcl-2 family members Bax and Bak induce mitochondrial outer membrane permeabilization (MOMP) to mediate cell death. Recently, it was shown that Bax and Bak are also required for mitochondrial permeability transition pore (MPTP)-dependent necrosis, where, in their non-oligomeric state, they enhance permeability characteristics of the outer mitochondrial membrane. Necroptosis is another form of regulated necrosis involving the death receptors and receptor interacting protein kinases (RIP proteins, by Ripk genes). Here, we show cells or mice deficient for Bax/Bak or cyclophilin D, a protein that regulates MPTP opening, are resistant to cell death induced by necroptotic mediators. We show that Bax/Bak oligomerization is required for necroptotic cell death and that this oligomerization reinforces MPTP opening. Mechanistically, we observe mixed lineage kinase domain-like (MLKL) protein and cofilin-1 translocation to the mitochondria following necroptosis induction, while expression of the mitochondrial matrix isoform of the antiapoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), is significantly reduced. Some of these effects are lost with necroptosis inhibition in Bax/Bak1 double , Ppif-/-, or Ripk3-/- fibroblasts. Hence, downstream mechanisms of cell death induced by necroptotic stimuli utilize both Bax/Bak to generate apoptotic pores in the outer mitochondrial membrane as well as MPTP opening in association with known mitochondrial death modifying proteins.

No MeSH data available.


Related in: MedlinePlus

Upstream necroptotic regulators signal to the mitochondrion through Mcl-1.A, Western blots for Bax, Bak, BID, Bcl-2, Mcl-1, MLKL, RIP3, cofilin-1, CypD, Complex V and III (mitochondrial loading control), and αtubulin (cytoplasmic loading control) from cytoplasmic and mitochondrial extracts from WT, Ripk3 , Bax/Bak1 DKO, and Ppif  MEFs treated with TNFα and zVAD-FMK (TZ) or vehicle (Veh.) for 6 hours. B, Immunocytochemistry for MLKL (green) and mitochondrial protein SAMM50 (red) on WT MEFs treated with TNFα and zVAD-FMK (TZ) or vehicle (Veh.) for 3 hours. Nuclei are stained with Dapi (blue).
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pone.0130520.g004: Upstream necroptotic regulators signal to the mitochondrion through Mcl-1.A, Western blots for Bax, Bak, BID, Bcl-2, Mcl-1, MLKL, RIP3, cofilin-1, CypD, Complex V and III (mitochondrial loading control), and αtubulin (cytoplasmic loading control) from cytoplasmic and mitochondrial extracts from WT, Ripk3 , Bax/Bak1 DKO, and Ppif MEFs treated with TNFα and zVAD-FMK (TZ) or vehicle (Veh.) for 6 hours. B, Immunocytochemistry for MLKL (green) and mitochondrial protein SAMM50 (red) on WT MEFs treated with TNFα and zVAD-FMK (TZ) or vehicle (Veh.) for 3 hours. Nuclei are stained with Dapi (blue).

Mentions: It is known that RIP1, RIP3 and MLKL are the integral regulators of necroptotic cell death but it is unclear how these effectors might connect downstream to the MOMP or the MPTP. To address this issue we isolated mitochondria from WT, Ripk3 , Bax/Bak1 DKO and Ppif MEFs treated with and without TNFα and zVAD-fmk and probed for known regulators of necroptosis and the MOMP and MPTP (Fig 4A). We found that upon treatment of TNFα and zVAD-fmk the expression of MLKL increases in the mitochondrial fraction (Fig 4A). To confirm this result we performed immunocytochemistry for MLKL and SAMM50 (mitochondrial marker), which showed greater translocation to the mitochondria in WT MEFs treated with TNFα and zVAD-fmk compared with vehicle stimulation (Fig 4B). However, necroptosis-induced translocation of MLKL to the mitochondria was not observed in the Ripk3-/- MEFs suggesting that this event required RIP3 activity (Fig 4A). Less MLKL translocation to the mitochondria was also observed in Bax/Bak1 DKO MEFs with TNFα and zVAD-fmk (Fig 4A).


Necroptosis Interfaces with MOMP and the MPTP in Mediating Cell Death.

Karch J, Kanisicak O, Brody MJ, Sargent MA, Michael DM, Molkentin JD - PLoS ONE (2015)

Upstream necroptotic regulators signal to the mitochondrion through Mcl-1.A, Western blots for Bax, Bak, BID, Bcl-2, Mcl-1, MLKL, RIP3, cofilin-1, CypD, Complex V and III (mitochondrial loading control), and αtubulin (cytoplasmic loading control) from cytoplasmic and mitochondrial extracts from WT, Ripk3 , Bax/Bak1 DKO, and Ppif  MEFs treated with TNFα and zVAD-FMK (TZ) or vehicle (Veh.) for 6 hours. B, Immunocytochemistry for MLKL (green) and mitochondrial protein SAMM50 (red) on WT MEFs treated with TNFα and zVAD-FMK (TZ) or vehicle (Veh.) for 3 hours. Nuclei are stained with Dapi (blue).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465034&req=5

pone.0130520.g004: Upstream necroptotic regulators signal to the mitochondrion through Mcl-1.A, Western blots for Bax, Bak, BID, Bcl-2, Mcl-1, MLKL, RIP3, cofilin-1, CypD, Complex V and III (mitochondrial loading control), and αtubulin (cytoplasmic loading control) from cytoplasmic and mitochondrial extracts from WT, Ripk3 , Bax/Bak1 DKO, and Ppif MEFs treated with TNFα and zVAD-FMK (TZ) or vehicle (Veh.) for 6 hours. B, Immunocytochemistry for MLKL (green) and mitochondrial protein SAMM50 (red) on WT MEFs treated with TNFα and zVAD-FMK (TZ) or vehicle (Veh.) for 3 hours. Nuclei are stained with Dapi (blue).
Mentions: It is known that RIP1, RIP3 and MLKL are the integral regulators of necroptotic cell death but it is unclear how these effectors might connect downstream to the MOMP or the MPTP. To address this issue we isolated mitochondria from WT, Ripk3 , Bax/Bak1 DKO and Ppif MEFs treated with and without TNFα and zVAD-fmk and probed for known regulators of necroptosis and the MOMP and MPTP (Fig 4A). We found that upon treatment of TNFα and zVAD-fmk the expression of MLKL increases in the mitochondrial fraction (Fig 4A). To confirm this result we performed immunocytochemistry for MLKL and SAMM50 (mitochondrial marker), which showed greater translocation to the mitochondria in WT MEFs treated with TNFα and zVAD-fmk compared with vehicle stimulation (Fig 4B). However, necroptosis-induced translocation of MLKL to the mitochondria was not observed in the Ripk3-/- MEFs suggesting that this event required RIP3 activity (Fig 4A). Less MLKL translocation to the mitochondria was also observed in Bax/Bak1 DKO MEFs with TNFα and zVAD-fmk (Fig 4A).

Bottom Line: Mechanistically, we observe mixed lineage kinase domain-like (MLKL) protein and cofilin-1 translocation to the mitochondria following necroptosis induction, while expression of the mitochondrial matrix isoform of the antiapoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), is significantly reduced.Some of these effects are lost with necroptosis inhibition in Bax/Bak1 double , Ppif-/-, or Ripk3-/- fibroblasts.Hence, downstream mechanisms of cell death induced by necroptotic stimuli utilize both Bax/Bak to generate apoptotic pores in the outer mitochondrial membrane as well as MPTP opening in association with known mitochondrial death modifying proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, United States of America.

ABSTRACT
During apoptosis the pro-death Bcl-2 family members Bax and Bak induce mitochondrial outer membrane permeabilization (MOMP) to mediate cell death. Recently, it was shown that Bax and Bak are also required for mitochondrial permeability transition pore (MPTP)-dependent necrosis, where, in their non-oligomeric state, they enhance permeability characteristics of the outer mitochondrial membrane. Necroptosis is another form of regulated necrosis involving the death receptors and receptor interacting protein kinases (RIP proteins, by Ripk genes). Here, we show cells or mice deficient for Bax/Bak or cyclophilin D, a protein that regulates MPTP opening, are resistant to cell death induced by necroptotic mediators. We show that Bax/Bak oligomerization is required for necroptotic cell death and that this oligomerization reinforces MPTP opening. Mechanistically, we observe mixed lineage kinase domain-like (MLKL) protein and cofilin-1 translocation to the mitochondria following necroptosis induction, while expression of the mitochondrial matrix isoform of the antiapoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), is significantly reduced. Some of these effects are lost with necroptosis inhibition in Bax/Bak1 double , Ppif-/-, or Ripk3-/- fibroblasts. Hence, downstream mechanisms of cell death induced by necroptotic stimuli utilize both Bax/Bak to generate apoptotic pores in the outer mitochondrial membrane as well as MPTP opening in association with known mitochondrial death modifying proteins.

No MeSH data available.


Related in: MedlinePlus