Necroptosis induced by RIPK3 requires MLKL but not Drp1

Abstract

Necroptosis is a mechanism by which cells can kill themselves that does not require caspase activity or the presence of the pro-apoptotic Bcl-2 family members Bax or Bak. It has been reported that RIPK3 (receptor interacting protein kinase 3) activates MLKL (mixed lineage kinase domain-like) to cause cell death that requires dynamin-related protein 1 (Drp1), because survival was increased in cells depleted of Drp1 or treated with the Drp1 inhibitor mdivi-1. To analyze necroptosis in a system that does not require addition of tumor necrosis factor (TNF), we used a construct that allows RIPK3 to be induced in cells, and then dimerized via an E. coli gyrase domain fused to its carboxyl-terminus, using the dimeric gyrase binding antibiotic coumermycin. We have previously shown elsewhere that RIPK3 dimerized in this manner not only induces necroptosis but also apoptosis, which can be inhibited by the broad-spectrum caspase inhibitor Q-VD-OPh (QVD). In response to RIPK3 dimerization, wild-type mouse embryonic fibroblasts (MEFs) underwent cell death that was reduced but not completely blocked by QVD. In contrast, death upon dimerization of RIPK3 in Mlkl(-/-) MEFs was completely inhibited with QVD, confirming that MLKL is required for necroptosis. Similar to wild-type MEFs, most Drp1(-/-) MEFs died when RIPK3 was activated, even in the presence of QVD. Furthermore, overexpression of wild-type MLKL or dominant active mutants of MLKL (Q343A or S345E/S347E) caused death of wild-type and Drp1(-/-) MEFs that was not inhibited with QVD. These results indicate that necroptosis caused by RIPK3 requires MLKL but not Drp1.

Figure 1

Drp1 and MLKL are not required for cell death induced by TNF plus smac-mimetic. (a) WT, Mlkl^−/−, and Drp1^−/− MEFs were harvested, and lysates resolved on replicate gels were probed for MLKL, Drp1, or RIPK3. β-Actin was used as a loading control. (b–d) WT, Mlkl^−/−, and Drp1^−/− MEFs were treated with 100 ng/ml TNF and/or 500 nM smac-mimetic for 24, 48, or 72 h. Cells were then stained with propidium iodide (PI) and analyzed by flow cytometry to detect loss of plasma membrane integrity. Error bars are S.E.M., where n=3 independently performed experiments (repeat 1=square, repeat 2= triangle, repeat 3= circle). (e) Drp1^−/− MEFs were infected with a lentiviral vector expressing FLAG-Drp1. Cells were induced with 1 μg/ml doxycycline for 24 h, harvested, and lysates run on replicate gels were probed for FLAG or β-actin as a loading control. (f) Drp1^−/− MEFs were treated with 1 μg/ml doxycycline and/or 100 ng/ml TNF and 500 nM smac-mimetic for 24 h. Cells were stained with PI and analyzed by flow cytometry. Error bars are S.E.M., where n=3 independently performed experiments (repeat 1=square, repeat 2= triangle, repeat 3= circle)

Figure 2

Necroptosis induced by RIPK3 dimerization occurs independently of Drp1. (a) WT, Mlkl^−/−, and Drp1^−/− MEFs were infected with a lentiviral vector expressing FLAG-tagged RIPK3-gyrase. Cells were induced with 10 nM 4HT for 24 h, harvested, and lysates on replicate gels were probed for FLAG or β-actin as a loading control. (b) Where indicated, cells were pretreated with 10 μM QVD for 1 h and subsequently treated with 10 nM 4HT and/or 700 nM coumermycin for 24 h. Cells were stained with PI and cell viability determined by flow cytometry. Error bars are S.E.M., where n=3 independently performed experiments (repeat 1=square, repeat 2= triangle, repeat 3=circle). (c) Inhibition of the GTPase domain of Drp1 does not protect from RIPK3 dimerization-induced cell death. WT MEFs expressing 4HT-inducible RIPK3-gyrase were pretreated with 100 μM of the Drp1 inhibitor mdivi-1 for 1 h, followed by treatment with 10 nM 4HT and 700 nM coumermycin for 24 h. Cells were stained with PI and analyzed by flow cytometry. Error bars are S.E.M., where n=3 independently performed experiments (repeat 1=square, repeat 2= triangle, repeat 3= circle). (d) WT MEFs expressing 4HT-inducible RIPK3-gyrase were pretreated with 100 μM mdivi-1 for 1 h, followed by treatment with 10 nM 4HT and 700 nM coumermycin for 24 h. Cells were resuspended using trypsin, replated, and after 5 days stained with crystal violet to assess clonogenicity. Inhibition of Drp1 by mdivi-1 was not able to protect clone-forming cells from death

Figure 3

Overexpression of MLKL induces cell death of WT and Drp1^−/− MEFs. (a) WT and Drp1^−/− MEFs were infected with a lentiviral vector expressing WT MLKL or a dominant active MLKL (Q343A). Cells were treated with 1 μg/ml doxycycline for 24 h to induce MLKL expression. Lysates were harvested, run on replicate gels, and probed for MLKL or β-actin as a loading control. (b) WT MEFs, where indicated, were pretreated with 10 μM QVD for 1 h and subsequently treated with 1 μg/ml doxycycline for 24 h. Cells were stained with propidium iodide (PI) and analyzed by flow cytometry. Error bars are S.E.M, where n=3 independently performed experiments (repeat 1=square, repeat 2= triangle, repeat 3=circle). (c and d) Where indicated, Drp1^−/− MEFs were pretreated with 10 μM QVD for 1 h and subsequently treated with 1 μg/ml doxycycline (to induce MLKL expression) and/or 10 nM 4HT (to induce RIPK3-gyrase expression) for 24 h. Cells were stained with PI and analyzed by flow cytometry. Error bars are S.E.M., where n=3 independently performed experiments (repeat 1=square, repeat 2= triangle, repeat 3= circle). (e) WT and Drp1^−/− MEFs were infected with a lentiviral vector bearing dominant active (SSEE) mutant MLKL. Cells were treated with 1 μg/ml doxycycline to induce SSEE MLKL expression. Lysates were harvested, run on replicate gels, and probed for MLKL or β-actin as a loading control. (f) Where indicated, WT and Drp1^−/− MEFs were pretreated with 10 μM QVD for 1 h and subsequently treated with 1 μg/ml doxycycline for 24 h. Cells were stained with PI and analyzed by flow cytometry. Error bars are S.E.M., where n=3 independently performed experiments (repeat 1=square, repeat 2= triangle, repeat 3= circle). (g) Representative contour plots of cell death induced by MLKL (SSEE) in WT and Drp1^−/− MEFs. Where indicated, WT and Drp1^−/− MEFs were pretreated with 10 μM QVD for 1 h and subsequently treated with 1 μg/ml doxycycline for 24 h. Cells were stained with PI and analyzed by flow cytometry. Note the increase in PI-staining cells when MLKL (SSEE) is induced in both WT and Drp1^−/− MEFs, and this is not reduced by pretreatment with QVD