RIPK1- and RIPK3-induced cell death mode is determined by target availability

Abstract

Both receptor-interacting protein kinase 1 (RIPK1) and RIPK3 can signal cell death following death receptor ligation. To study the requirements for RIPK-triggered cell death in the absence of death receptor signaling, we engineered inducible versions of RIPK1 and RIPK3 that can be activated by dimerization with the antibiotic coumermycin. In the absence of TNF or other death ligands, expression and dimerization of RIPK1 was sufficient to cause cell death by caspase- or RIPK3-dependent mechanisms. Dimerized RIPK3 induced cell death by an MLKL-dependent mechanism but, surprisingly, also induced death mediated by FADD, caspase 8 and RIPK1. Catalytically active RIPK3 kinase domains were essential for MLKL-dependent but not for caspase 8-dependent death. When RIPK1 or RIPK3 proteins were dimerized, the mode of cell death was determined by the availability of downstream molecules such as FADD, caspase 8 and MLKL. These observations imply that rather than a 'switch' operating between the two modes of cell death, the final mechanism depends on levels of the respective signaling and effector proteins.

Figure 1

Expression and dimerization of RIPK1 gyrase and RIPK3 gyrase is sufficient to cause death of MEFs. (a) FLAG-tagged RIPK1 gyrase B and FLAG-tagged RIPK3 gyrase B constructs. Inducible expression is driven by binding of a GAL4-T2 estrogen Receptor-VP16 transcription factor to 5 upstream UAS elements in the presence of 4HT. The divalent antibiotic coumermycin (C) dimerizes the gyrase B domains. (b) Western blots of lysates from MEFs bearing the inducible RIPK1 gyrase or RIPK3 gyrase constructs in the absence or presence (+) of 16 nM 4HT for 6 h were probed with antibodies to RIPK1 or RIPK3. The same blots were probed with antibodies to β-actin as a loading control, either simultaneously, in the case of the RIPK1 blot, or sequentially in the case of the RIPK3 blot. Endogenous and induced fusion proteins are labeled. (c) Analysis of cell viability by propidium iodide exclusion in untreated cells, and those treated for 24 h with 800 nM C alone, 16 nM 4HT alone or 4HT+C. PI-negative (viable) cells are boxed, and their percentages are indicated. Although induction of RIPK1 gyrase alone caused some cell death (depending on expression levels), addition of coumermycin greatly increased the death of cells expressing either the RIPK1 gyrase or RIPK3 gyrase proteins

Figure 2

MEF lines and L929 cells express varying amounts of cell death proteins. (a) Lysates were prepared from WT and gene-deleted MEF lines as well as L929 mouse fibroblastoid cells, and identical aliquots were run on three replicate gels and, after blotting, were probed with antibodies to the indicated proteins. The gene-deleted lines lacked expression of the corresponding proteins, but, in addition, the Fadd^−/− MEFs expressed barely detectable levels of MLKL, the Ripk1^−/− line 3.6 expressed very low levels of MLKL, the Ripk3^−/− line had low levels of RIPK1 and the caspase 8^−/− line did not express detectable levels of RIPK3. (b) TNF alone causes death of L929 cells, strongly inhibited by nec-1 but unaffected by QVD. Death of Mlkl^−/− MEFs caused by dimerization of RIPK3 gyrase is strongly inhibited by QVD but not by nec-1. L929 cells were treated with 100 ng/ml human Fc-TNF and Mlkl^−/− cells were treated with 16 nM 4HT and 800 nM C for 24 h in the presence or absence of 50 μM nec-1 or 10 μM QVD. Viability was determined by PI exclusion on a flow cytometer. Triangles and circles indicate independently performed experiments, and error bars show S.E.M. values

Figure 3

Dimerization of RIPK1 can induce death of L929 cells by a caspase-independent mechanism and death of MEFs by both caspase-dependent and caspase-independent mechanisms. (a) Various MEF lines and L929 cells bearing the inducible RIPK1 gyrase construct were either untreated or treated with 4HT for 6 h. Lysates were separated by SDS-PAGE, blotted and probed with antibodies to RIPK1 and β-actin as a loading control. Low MW bands in lanes from treated cells are assumed to be breakdown products of the RIPK1 gyrase resulting from incomplete inhibition of proteases during cell lysis. (b) Fibroblast lines bearing the inducible RIPK1 gyrase construct were either untreated or treated with 4HT, 4HT+C or 4HT+C after pretreatment for 1 h with 10 μM QVD to inhibit caspase activity. After a further 24 h, viability of the cells was determined by PI exclusion. Mean±S.E.M. are shown for three independent sets of experiments. The levels of endogenous RIPK3 and caspase 8 (as determined by western blot; see Figure 2a) in each line are indicated below the histograms. The mechanism of cell death was inferred from the protection given by caspase inhibition with QVD, and caspase-independent cell death was presumed to be necroptosis. (c) In L929 cells, death induced by expression of RIPK1 gyrase was not inhibited by QVD, but was reduced by nec-1, and hence is mainly necroptotic. When RIPK1 gyrase was dimerized by coumermycin, inhibition of cell death by nec-1 was much less effective. The percentage of PI-negative (viable) cells is shown in the boxes

Figure 4

RIPK3 gyrase can cause cell death by a mechanism that requires neither caspase 8 nor RIPK1 (necroptosis). (a) Western blot analysis of lysates from caspase 8^−/− MEFs bearing the inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT for 6 h. The same blots were probed with antibodies to RIPK3, and reprobed with antibodies to β-actin as a loading control. (b) Caspase 8^−/− MEFs bearing the inducible FLAG-RIPK3 gyrase construct were either untreated or treated with combinations of 16 nM 4HT and 800 nM C, with or without 10 μM QVD, for 24 h. Viability of the cells was determined by PI exclusion. Mean±S.E.M. of five independently performed experiments are shown. Predictably, QVD was unable to prevent death of the caspase 8^−/− MEFs, indicating that RIPK3 was activating another death mechanism, presumably necroptosis. (c) Western blot of lysates of L929 cells bearing the inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT for 6 h. The same blots were probed with antibodies to RIPK3, and reprobed with antibodies to β-actin as a loading control. (d) L929 cells bearing the inducible FLAG-RIPK3 gyrase construct were either untreated or treated with combinations of 16 nM 4HT, 800 nM C, 10 μM QVD and 100 ng/ml TNF with or without 50 μM nec-1 for 24 h, and cell viability was determined by PI exclusion. Mean±S.E.M. of five independently performed experiments are shown. Dimerized RIPK3 caused death of the L929 cells, and this was not inhibited by QVD, or by the same doses of nec-1 that were able to block necroptosis induced by TNF. (e) Western blot analysis of lysates from Fadd^−/− MEFs bearing a 4HT-inducible RIPK3 gyrase construct and a doxycycline (dox)-inducible MLKL construct in the absence or presence (+) of 16 nM 4HT with or without 100 ng/ml dox for 6 h. The same blot was probed sequentially with antibodies to RIPK3, MLKL and β-actin as a loading control. Note that these FADD ^−/− MEFs do not express detectable levels of endogenous MLKL. (f) Fadd^−/− MEFs were either untreated or treated with combinations of 16 nM 4HT, 800 nM C and 100 ng/ml dox, with or without 10 μM QVD, for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of six independently performed experiments are shown. Dimerized RIPK3 was not able to cause death of the cells unless MLKL was also induced, and the death that occurred was not inhibited by QVD, and was therefore presumably necroptosis. (g) Western blot analysis of lysates from a Ripk1^−/− MEF line (3.6) bearing a 4HT-inducible RIPK3 gyrase construct and a dox-inducible MLKL construct in the absence or presence (+) of 16 nM 4HT with or without 100 ng/ml dox for 6 h. The same blot was probed sequentially with antibodies to MLKL, RIPK3 and β-actin as a loading control. (h) Ripk1^−/− 3.6 MEFs were either untreated or treated with combinations of 16 nM 4HT, 800 nM C and 100 ng/ml dox, with or without 10 μM QVD, for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of three independently performed experiments are shown. Dimerized RIPK3 was able to cause some death of the Ripk1^−/− cells, whereas MLKL alone had no effect. When MLKL and RIPK3 dimers were induced together, more cell death occurred, and this was not blocked by QVD

Figure 5

Activation of RIPK3 can trigger caspase-dependent apoptosis as well as MLKL-dependent necroptosis. (a) Western blot analysis of lysates from WT and Ripk1^−/− 1.7 MEF lines bearing a 4HT-inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT for 6 h. The same blots were probed with antibodies to RIPK3 and reprobed with antibodies to β-actin as a loading control. (b) WT (dark gray columns; data from three independent cell lines) and Ripk1^−/− MEFs (light gray columns; data combined from lines 1.7 and 2.6) were either untreated or treated with combinations of 16 nM 4HT, 800 nM C, 50 μM nec-1 and/or 10 μM QVD for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of 3–5 independently performed experiments are shown. RIPK3 was able to cause death of both WT and Ripk1^−/− cells, but it caused more cell death in the presence of RIPK1. In neither case was the death affected by nec-1, but it was reduced by QVD, indicating that RIPK3 can trigger a caspase-dependent death process, but it does so more efficiently in cells expressing RIPK1. (c) Western blot analysis of lysates from Mlkl^−/− MEFs bearing a 4HT-inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT for 6 h. The same blots were probed with antibodies to RIPK3 and reprobed with antibodies to β-actin as a loading control. (d) Mlkl^−/− MEFs were either untreated or treated with combinations of 16 nM 4HT and 800 nM C, with or without 50 μM nec-1 and/or 10 μM QVD, for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of nine independently performed experiments are shown. RIPK3 was able to cause death of Mlkl^−/− cells that was completely blocked by QVD, indicating that in the absence of MLKL, activated RIPK3 causes only caspase-dependent cell death. (e) WT and Ripk1^−/− MEFs bearing the RIPK3 gyrase construct were treated with 10 nM 4HT for 24 h and/or 700 nM C for 2 h. Lysates were run on replicate blots and were probed with antibodies to full-length caspase 8, processed caspase 8, processed caspase 3 and PARP. The full-length caspase 8 blots were washed and re-probed for β-actin to act as a loading control. (f) Western blot analysis of lysates from L929 cells and WT MEF lines bearing a 4HT-inducible RIPK3 gyrase construct in the absence or presence (+) of 16 nM 4HT with or without 800 nM C for 5 h. The same blots were probed sequentially with antibodies to PARP, pro-caspase 8 and β-actin as a loading control. Dimerized RIPK3 gyrase can activate caspase 8 (but not detectably in L929 cells) and lead to processing of downstream caspases and PARP, and it does so more efficiently in the presence of RIPK1

Figure 6

In the absence of both caspase 8 and MLKL, activated RIPK3 fails to induce death. (a) Western blot analysis of lysates from WT, caspase 8^−/−Ripk3^−/− and caspase 8^−/−Mlkl^−/− mouse dermal fibroblasts (MDFs) either untreated or treated (+) with 10 nM 4HT for 24 h. The blots were probed for FLAG followed by β-actin as a loading control. (b) WT, caspase 8^−/−Ripk3^−/− and caspase 8^−/−Mlkl^−/− MDFs were either untreated or treated with combinations of 10 nM 4HT, 700 nM C and 10 μM QVD for 24 h. Viability was determined by PI exclusion. Mean±S.E.M. of three independently performed experiments are shown. Dimerized RIPK3 was able to cause some death of the WT cells, and this could be partly blocked by QVD. When both caspase 8 and MLKL were absent, no death was induced

Figure 7

K51A mutant of RIPK3 gyrase is catalytically inactive but apparently unstable. (a) WT and K51A RIPK3 gyrase were immunoprecipitated using anti-FLAG coupled beads from cells expressing the respective constructs, and used in in vitro kinase assays to show whether they were able to auto-phosphorylate. Washed beads were resuspended in 30 μl of kinase buffer (20 mM HEPES, pH 7.5, 10 mM MnCl₂ and 1 mM DTT), divided into two aliquots that were incubated in the presence versus absence of 250 μM ATP at 30°C for 20 min and analyzed by electrophoresis followed by replicate western blots that were probed with anti-phosphothreonine and anti-RIPK3. (b) WT, Mlkl^−/−, Ripk3^−/−, caspase 8^−/− MEFs and L929 cells bearing WT or K51A (kinase dead) RIPK3 gyrase constructs were either untreated or treated with combinations of 16 nM 4HT, 800 nM C and 10 μM QVD for 16 h. Viability was determined by PI exclusion. Mean±S.E.M. of three independently performed experiments are shown. The kinase-mutant K51A RIPK3 was unable to cause statistically significant levels of cell death in the apoptosis-prone Mlkl^−/−, or in caspase 8^−/− cells (that lack detectable RIPK3). It appeared to induce death of a small number of WT and Ripk3^−/− cells, but it was only able to cause convincing levels of cell death in L929 cells that express endogenous RIPK3 and show reduced activation of caspase 8. The death in L929 cells could not be blocked by QVD, and hence it is presumed to be necroptosis. Note the low levels of expression of K51A relative to WT in (a), (c) and track 6 of (d). (c) Western blots of lysates from MEFs bearing WT or K51A (kinase dead) RIPK3 gyrase constructs that were untreated or treated with 16 nM 4HT and 700 nM C for 6 h were probed sequentially with antibodies to caspase 8, PARP and RIPK3, followed by β-actin as loading controls. The asterisk indicates a nonspecific band. (d) Western blots of lysates from WT, Mlkl^−/−, Ripk3^−/−, caspase 8^−/− MEFs and L929 cells bearing the K51A (kinase dead) RIPK3 gyrase construct, and WT and L929 cells bearing the WT RIPK3 gyrase were untreated or treated with 16 nM 4HT for 6 h and probed with antibodies to RIPK3 followed by β-actin as loading controls

Figure 8

The kinase activity of RIPK3 is needed for it to cause necrosis but not apoptosis. (a and b) Recombinant D143N and R142G mutant mRIPK3 kinase domains are catalytically defective. (a) A total volume of 1 μg of wild-type (WT), D143N and R142G mRIPK3 kinase domain, which had been expressed in insect cells and purified, was resolved by reducing SDS-PAGE and stained with Coomassie Blue. Comparable intensity of Coomassie-stained bands confirmed reliability of concentration estimation by Absorbance at 280 nm. These concentration estimates were used subsequently to ensure that comparable quantities of WT and mutant RIPK3 were assayed in in vitro kinase assays. (b) In vitro kinase assays demonstrate that WT, but not D143N, mRIPK3 can autophosphorylate and phosphorylate recombinant MLKL, consistent with our prior characterization of recombinant D143N mRIPK3, whereas R142G mRIPK3 exhibits defective catalytic activity in autophosphorylation and MLKL phosphorylation. The shown experiment is representative of two independent experiments. BSA was added as a carrier protein (100 ng/μl). The Coomassie-stained image of the representative gel is shown on the left and the autoradiograph of the same gel on the right. Only 25 ng of each recombinant RIPK3 kinase domain was loaded in each experiment and is consequently not visible by Coomassie Blue staining. (c) Western blots of lysates from MEFs bearing WT, D143N (kinase dead) and R142G (kinase compromised) RIPK3 gyrase constructs that were untreated or treated with 16 nM 4HT and 800 nM C for 6 h were probed sequentially with antibodies to PARP and RIPK3, followed by β-actin as loading controls. The asterisks indicate nonspecific bands. Although a background level of cleaved PARP is detectable in all cell types in this experiment, the band intensity increases with induction of expression and dimerization of RIPK3 gyrase only in Ripk3^−/− and Mlkl^−/− cells, in which caspase-dependent apoptosis occurs (see f and g). (d–g) Kinase-inactive or -compromised RIPK3 gyrase can induce apoptosis, but cannot induce necroptosis without the presence of (endogenous) WT RIPK3. (d) Caspase 8^−/− cells bearing the inducible RIPK3 gyrase construct were either untreated or treated with combinations of 16 nM 4HT, 800 nM C and 10 μM QVD. Mean±S.E.M. of four independently performed experiments are shown. (e) In the presence of WT RIPK3, kinase-inactive RIPK3 gyrase induces necroptosis. L929 cells bearing the inducible RIPK3-gyrase construct were either untreated or treated as in (d), or with the addition of nec-1. Mean±S.E.M. of four independently performed experiments are shown. The R142G mutant was as effective as the WT, and even the D143N mutant induced some cell death. (f) Apoptosis induced by dimerized RIPK3 is independent of its kinase activity. Mlkl^−/− cells bearing the respective RIPK3 gyrase constructs were untreated or treated as in (e). Mean±S.E.M. of three independently performed experiments are shown. Both R142G and D143N mutants caused the death of substantial numbers of cells, almost equivalent to WT. (g) Apoptosis induced by dimerized RIPK3 is independent of bystander RIPK3. Ripk3^−/− MEFs bearing the respective RIPK3 gyrase constructs were untreated or treated as in (e). Mean±S.E.M. of three independently performed experiments are shown. Again, cell death was substantial in response to the WT and each of the mutants

Figure 9

RIPK dimers are at the fork in the cell death pathway. (a) Depending on the availability of targets, forcibly dimerized RIPK1 recruits FADD and caspase 8 to induce apoptosis, or if autophosphorylation occurs, it recruits and phosphorylates RIPK3 that in turn phosphorylates MLKL to induce necroptosis. There is a slight bias toward apoptosis. (b) Similarly, forcibly dimerized RIPK3 recruits FADD and therefore caspase 8, but this is inefficient unless RIPK1 is available. If autophosphorylation occurs in the RIPK3 dimer, it phosphorylates MLKL and necroptosis follows. We predict that in normal cells responding to death signals, RHIM mediated homo- and hetero-dimerization of RIPK1 and RIPK3 mimics these activities. In the case of RIPK1, the dimerization can be via its DD