Abstract
RIP1 is an essential regulator of TNF-induced signaling complexes mediating NF-κB activation, apoptosis and necroptosis. Loss of Rip1 rescues the embryonic lethality of Fadd or Caspase-8-deficient mice, even though the double knockout mice die shortly after birth like Rip1-deficient mice. Recent studies demonstrated that mice expressing RIP1 kinase-dead mutants developed normally and resisted necroptotic stimuli in vitro and in vivo. However, the impact of RIP1 kinase activity on Fadd−/− embryonic development remains unknown. Here, we engineered two RIP1 kinase inactive mutant mouse lines, a Rip1K45A/K45A mouse line as previously reported and a novel Rip1Δ/Δ mouse line with an altered P-loop in the kinase domain. While RIP1K45A could not rescue the embryonic lethality of Fadd-deficient mice at E11.5, RIP1Δ rescued lethality of Fadd−/− mice at E11.5 and Fadd−/−Rip1Δ/Δ mice eventually died at E16.5 due to excessive death of fetal liver cells and unregulated inflammation. Under necropotosis-inducing conditions, comparing to Rip1K45A/K45A cells, Rip1Δ/Δcells displayed reduced phosphorylation and oligomerization of RIP3 and MLKL, which lead to increased cell viability. Thus, our study provides genetic evidence that different kinase inactive mutations have distinct impacts on the embryogenesis of Fadd-deficient mice, which might attribute to their extents of protection on necroptosis signaling.
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Main
The receptor-interacting protein kinase 1 (RIP1) functions as a key regulator for NF-κB activation, apoptosis and necroptosis induced by tumor necrosis factor (TNF-α).1, TNF-α and z-VAD were purchased from R&D (Minneapolis, MN, USA) and Calbiochem (Anaheim, CA, USA), respectively. The Smac mimetic, Cycloheximide and LPS were obtained from Sigma. Nec-1 was from Enzo Life Science (Alexis, USA). The following antibodies were used for western blotting: p-IkBa (Cell Signaling), p-ERK (Cell Signaling Technology, Danvers, MA, USA), ERK (Cell Signaling), p-P38 (Cell Signaling), P38 (Cell Signaling), p-P65 (Cell Signaling), p-JNK (Cell Signaling), JNK (Cell Signaling), PARP (Cell Signaling), Caspase-3 (Cell Signaling), HA (Cell Signaling), Caspase-8 (Enzo Life Science), RIP1 (BD Biosciences, Franklin Lakes, NJ, USA), p-RIP1(S166) (Cell Signaling) mouse RIP3 (ProSci, San Diego, CA, USA), MLKL (Abcam, Cambridge, UK), p-MLKL (Abcam), β-actin, α-Tubulin and anti-flag-HRP (Sigma). Anti-phospho-RIP3 antibody (mouse) was generated in our lab and immunoaffinity-purified. Cell viability was determined by measuring ATP levels using Cell Titer-Glo kit (Promega, Madison, WI, USA). Mice were housed in a specific pathogen-free facility, which belongs to Institute for Nutritional Sciences. Fadd−/− mice (C57BL/6) were gifted by Dr. Jianke Zhang (Thomas Jefferson University, Philadelphia, USA), and Rip3−/− mice (C57BL/6) were provided by Dr. **aodong Wang (NIBS, Bei**g, China). Animals were subsequently backcrossed on a C57BL/6 background for at least 10 generations. A novel mutant, RIP1Δ, was obtained from the experiment with two amino acids G26F27 in the P-loop of RIP1 deleted. To generate Rip1Δ/Δ, Rip1K45A/K45A, Rip1−/− mice by crispr-cas9 mutation system (Bioray Laboratories Inc., Shanghai, China), different sgRNA were designed to target RIP1 kinase domain. (Rip1Δ/Δmutant with gRNA: 5′-GACCTAGACAGCGGAGGCTT-3′; Rip1K45A/K45A mutant with gRNA: 5′-GCCCTGTGTATACTTTTTTC-3′; Rip1−/− with gRNA: 5′-GATGGCATCCAGTGACCTGC-3′). Additional information is provided upon request. All mutant mice and WT mice used in these studies shared a common genetic C57BL/6 background. Animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (CAS), University of Chinese Academy of Sciences. Male WT, Rip3−/− and Rip1Δ/Δ mice littermates at 8 weeks of age (n=7) were treated every hour for 12 consecutive hours with cerulein (50 μg/kg, Sigma) intraperitoneal injection. Animals were assayed 24 h after the first injection. Serum amylase activity was assayed by amylase activity assay kit (Sigma). MDFs were separated from the skin of newborn mice, and MEFs were isolated from E13.5 to E14.5 embryos. MDFs and MEFs were cultured in DMEM medium supplemented with 10% FBS and penicillin/streptomycin. BMDMs from isolated bone marrow cells collected from mouse femurs and tibias were induced to differentiate in vitro. Bone marrow cells were cultured for 7 days in RPMI medium containing 10% FBS, penicillin/streptomycin and 50 ng/ml M-CSF, and medium was changed every 2 days. Thymocytes were isolated from thymus and cultured in RPMI medium containing 10% FBS, penicillin/streptomycin and 50 μM β-ME. Cells were harvested at different time points, washed with PBS and lysates with 1 × SDS sample buffer containing 100 mM DTT and boiled for 5 min at 95 °C for reducing gel. For mouse tissue protein extraction, E14.5 fetal liver and other tissues were ground by pestle and mortar with liquid N2, and the protein was extracted with RIPA lysis buffer. The lysates were cleared by centrifugation for 20 min at 13 200 × g, quantified by BCA kit (Thermo Scientific, Rockford, IL, USA) then mixed with SDS sample buffer and boiled at 95 °C for 5 min. The samples were separated using SDS-PAGE, transferred to PVDF membrane (Millipore, Darmstadt, Germany) with 100 v for 2 h. The proteins were detected by using a chemiluminescent substrate (Thermo Scientific). To immunoprecipitate RIP1, cell extract protein was incubated for 3 h with 5 μl of RIP1 antibody (BD Biosciences). After mixing end over end for overnight (4 °C) with 30 μl of G-Agarose beads, the agarose was collected and washed three times with cell lysis buffer (Tris-HCl 20 mmol/l (pH 7.5), NaCl 150 mM, EDTA 1 mM, EGTA 1 mM, Triton X-100 1%, Sodium pyrophosphate 2.5 mM, β-Glycerrophosphate 1 mM, NaVO4 1 mM, Leupeptin 1 μg/ml.). Immunoprecipitates were denatured in SDS, subjected to SDS-PAGE, and immunoblotted. The cells were cultured in six-well plates and treated with indicated stimuli. Cells were harvested at different time points and lysed with 2 × DTT-free sample buffer (Tris-Cl (PH 6.8) 125 mM, SDS 4%, Glycerol 20%, Bromophenol blue 0.02%) immediately. Total cell lysates were separated using SDS-PAGE, and transferred to PVDF membrane (Millipore), and western blotting was performed with RIP3 or MLKL antibodies. Antibodies against mouse CD3, CD4, CD8, CD19, Mac-1 and Gr-1 from eBioscience (Carlsbad, CA, USA) were fluorescence-conjugated and were used for flow cytometry analysis in this study. We prepared single-cell suspension from lymph nodes, spleen and thymus, respectively, and stained them with fluorescence-conjugated antibodies for half an hour in staining buffer. After staining, cells were immediately analyzed by flow cytometry (FACSAria III, BD Biosciences). MDFs were plated overnight on coverslips before various stimulations. After stimulation, cells were washed with PBS and fixed with 4% PFA in PBS for 15 min. Next, the cells were blocked with 0.3% Triton X-100 and 5% normal donkey serum (Jackson immunoResearch, Baltimore Pike, West Grove, PA, USA) for 1 h at room temperature, then followed by first antibody incubation at 4 °C for overnight. Signals were developed with Alexa fluorescence antibodies (Invitrogen). Finally, the cells were stained with DAPI for 10 min. Confocal microscopy analysis was performed using a Zeiss 710 laser-scanning microscope (Zeiss, Thornwood, NY, USA). Total RNA was extracted using Trizol reagent (Life Technologies), according to the manufacturer’s instructions. After quantification, 2 μg total RNA was reverse transcribed to complementary DNA (Takara, Dalian, China). Transcript levels of indicated cytokines were quantified by quantitative RT-PCR on an ABI 7500 real-time PCR instrument with SYBR Green. Relative expression was calculated using LC32 as an internal control as indicated. Primers used were as follows: mIL-1b: 5′-CCCAACTGGTACATCAGCAC-3′ and 5′-TCTGCTCATTCACGAAAAGG-3′; mTNF: 5′-CCCACTCTGACCCCTTTACT-3′ and 5′-TTTGAGTCCTTGATGGTGGT-3′; mIL-6: 5′-CGGAGAGGAGACTTCACAGA-3′ and 5′-CCAGTTTGGTAGCATCCATC-3′; mCXCL-1: 5′-CTGGGATTCACCTCAAGAACATC-3′ and 5′-CAGGGTCAAGGCAAGCCTC-3′; mMCP-1: 5′-TTAAAAACCTGGATCGGAACCAA-3′ and 5′-GCATTAGCTTCAGATTTACGGGT-3′; mCCL-5:5′-GCTGCTTTGCCTACCTCTCC-3′ and 5′-TCGAGTGACAAACACGACTGC-3′.Materials and Methods
Reagents
Mice
Cerulein-induced acute pancreatitis
Isolation and culture of MDFs, MEFs, BMDMs and thymocytes
Immunoblotting and immunoprecipitation
RIP3 and MLKL oligomerization detection
Flow cytometry
Immunofluorescence
RT-PCR
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Acknowledgements
We thank Dr **aodong Wang (National Institute of Biological Sciences, Bei**g, China) for providing Ripk3−/− mice and Dr. Jianke Zhang (Thomas Jefferson University, Philadelphia, PA, USA) for providing Fadd+/− mice. We also thank Dr Yu Sun (David Geffen School of Medicine, UCLA, USA) for insightful discussions and critical reading of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (31571426) and the Ministry of Science and Technology of the People’s Republic of China (2016YFC1304900, 2016YFA0500100). HBZ was supported by Thousand Young Talents Program of the Chinese government.
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Liu, Y., Fan, C., Zhang, Y. et al. RIP1 kinase activity-dependent roles in embryonic development of Fadd-deficient mice. Cell Death Differ 24, 1459–1469 (2017). https://doi.org/10.1038/cdd.2017.78
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DOI: https://doi.org/10.1038/cdd.2017.78
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