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. 2020 May;27(5):1539-1553.
doi: 10.1038/s41418-019-0434-2. Epub 2019 Oct 28.

Caspase-8, receptor-interacting protein kinase 1 (RIPK1), and RIPK3 regulate retinoic acid-induced cell differentiation and necroptosis

Masataka Someda  1   2 , Shunsuke Kuroki  1   3 , Hitoshi Miyachi  4 , Makoto Tachibana  3 , Shin Yonehara  5   6   7
Affiliations

Caspase-8, receptor-interacting protein kinase 1 (RIPK1), and RIPK3 regulate retinoic acid-induced cell differentiation and necroptosis

Masataka Someda et al. Cell Death Differ. 2020 May.

Abstract

Among caspase family members, Caspase-8 is unique, with associated critical activities to induce and suppress death receptor-mediated apoptosis and necroptosis, respectively. Caspase-8 inhibits necroptosis by suppressing the function of receptor-interacting protein kinase 1 (RIPK1 or RIP1) and RIPK3 to activate mixed lineage kinase domain-like (MLKL) VSports手机版. Disruption of Caspase-8 expression causes embryonic lethality in mice, which is rescued by depletion of either Ripk3 or Mlkl, indicating that the embryonic lethality is caused by activation of necroptosis. Here, we show that knockdown of Caspase-8 expression in embryoid bodies derived from ES cells markedly enhances retinoic acid (RA)-induced cell differentiation and necroptosis, both of which are dependent on Ripk1 and Ripk3; however, the enhancement of RA-induced cell differentiation is independent of Mlkl and necrosome formation. RA treatment obviously enhanced the expression of RA-specific target genes having the retinoic acid response element (RARE) in their promoter regions to induce cell differentiation, and induced marked expression of RIPK1, RIPK3, and MLKL to stimulate necroptosis. Caspase-8 knockdown induced RIPK1 and RIPK3 to translocate into the nucleus and to form a complex with RA receptor (RAR), and RAR interacting with RIPK1 and RIPK3 showed much stronger binding activity to RARE than RAR without RIPK1 or RIPK3. In Caspase-8-deficient as well as Caspase-8- and Mlkl-deficient mouse embryos, the expression of RA-specific target genes was obviously enhanced. Thus, Caspase-8, RIPK1, and RIPK3 regulate RA-induced cell differentiation and necroptosis both in vitro and in vivo. .

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Knockdown of Casp8 expression in ES cells enhanced RA-induced cell differentiation. a Dox-inducible (Tet-On) shCasp8-expression system. TetR, tetracycline repressor; and U6tet, mouse U6 promoter joining the tetracycline operator. b Validation of induced knockdown of Casp8 expression in Tet-On shCasp8 ES cells by western blot analysis after treatment with 1 μg/ml Dox for indicated days. Molecular weight markers are indicated (kDa). Neuronal differentiation of Dox (1 μg/ml)-treated or -untreated Tet-On shCasp8 and Tet-On shGFP ES cells was analyzed by phase-contrast microscopy (c) or fluorescence microscopy (d) after 6-day formation of EBs. EBs were treated with or without 1 μM RA for last 4 days. Scale bars, 200 μm. Cells were stained with anti-Tuj1 antibody in d. e qRT-PCR analysis of Oct3/4 was carried out using EBs defined in c. f qRT-PCR analysis of Nestin, Tuj1, and Casp8 was carried out using ES cells at the indicated times after formation of EBs defined in c. **p < 0.01, *p < 0.05 and n.s.d. (no significant difference: p > 0.95)
Fig. 2
Fig. 2
Knockdown of Casp8 expression in ES cells markedly enhanced RA signaling dependently on RIPK3. a Tet-On shCasp8 and Tet-On shGFP ES cells were cultured for 4 days with or without 1 μg/ml Dox and then treated with or without 1 μM RA for 24 h in the presence or absence of Dox. Subsequently, expression levels of RA-specific target genes, Crabp2, Hoxb1, Cyp26a1, and Rarb, were analyzed by qRT-PCR. b Dual-luciferase assay for RARE was performed using ES cells defined in a. c Western blot analysis of RIPK3 expression was carried out in Tet-On shCasp8 ES cells expressing shLacZ or shRipk3 in the absence of Dox. Actin was detected as a control. Molecular weight markers are indicated (kDa). d qRT-PCR analysis of Nestin and Tuj1 expression was performed using EBs derived from Tet-On shCasp8 ES cells expressing shLacZ or shRipk3 after 6 days formation of EBs in the presence or absence of 1 μg/ml Dox. EBs were treated with 1 μM RA for last 4 days. e Dual-luciferase assay for RARE was carried out using Tet-On shCasp8 ES cells expressing shLacZ or shRipk3 after treatment with or without 1 μM RA for 24 h in the presence or absence of 1 μg/ml Dox. f, g Tet-On shCasp8 P19 cells expressing shLacZ or shRipk3 were infected with lentiviral vectors carrying 3xFlag-Wt Ripk3 or K51A Ripk3. These cells were cultured with or without 1 μg/ml Dox for 5 days, and then treated with or without 1 μM RA for 24 h in the presence or absence of Dox. Subsequently, western blot analysis for RIPK3 (f) and qRT-PCR analysis for RA-inducible genes (g) were carried out. Vector, an empty vector. **p < 0.01 and *p < 0.05
Fig. 3
Fig. 3
RIPK1 but not MLKL is involved in the activation of RA signaling. a Expression levels of Ripk1 were analyzed by qRT-PCR using Tet-On shCasp8 P19 cells expressing shLacZ or shRipk1. b P19 cells defined in a were cultured for 4 days with or without 1 μg/ml Dox and then treated with or without 1 μM RA for 24 h in the presence or absence of Dox. Subsequently, qRT-PCR analysis of RA-induced genes, Crabp2, Hoxb1, and Cyp26a1, was performed. c Expression levels of Mlkl were analyzed by qRT-PCR using Tet-On shCasp8 P19 cells expressing shLacZ or shMlkl. Two shRNAs targeting different nucleotide sequences in Mlkl (shMlkl #1 and shMlkl #2) were used. d P19 cells defined in c were analyzed by qRT-PCR as described in b. qRT-PCR analysis of TUJ1 and RARB, and CD11b and RARB was carried out using SK-N-SH cells (e) and HL60 cells (f), respectively, expressing shLacZ or shCASP8 after treatment with DMSO or 10 µM NSA for 48 h together with or without 1 μM RA for last 24 h in the presence of DMSO or 10 µM NSA. g qRT-PCR analysis of RA-induced genes, Crabp2, Hoxb1, and Cyp26a1, was performed using Dox (1 μg/ml)-treated or -untreated Tet-On shCasp8 P19 cells cultured with or without 1 μM RA for 24 h in the presence of DMSO or 30 μM Nec-1. **p < 0.01, *p < 0.05 and n.s.d. (no significant difference: p > 0.95)
Fig. 4
Fig. 4
Knockdown of Casp8 expression in ES cells enhanced RA-induced necroptosis in EBs. a Dox (1 μg/ml)-treated or -untreated Tet-On shCasp8 or Tet-On shGFP ES cells expressing or not expressing shRipk3 were analyzed by phase-contrast microscopy after 4 days formation of EBs. EBs were treated with or without 1 μM RA and 30 μM Nec-1 for last 2 days. Scale bars, 200 μm. b Relative diameters of EBs defined in a were measured under phase-contrast microscopy. n = 10. c Cell death was quantified by a lactate dehydrogenase (LDH) release assay after 4 days of formation of EBs derived from Tet-On shCasp8 ES cells expressing shRipk3 or shLacZ. EBs were treated with or without 1 μM RA and 30 μM Nec-1 for last 2 days. ES cells or EBs were cultured with 1 µg/ml Dox throughout the experiments. d Tet-On shCasp8 ES cells expressing shLacZ or both shCasp3 and shCasp7 were subjected to western blot analysis using anti-Caspase-3 or anti-Caspase-7 antibodies. Actin was detected as a control. e Tet-On shCasp8 ES cells expressing shLacZ or shMlkl #1 were subjected to qRT-PCR analysis for Mlkl. f Cell death was quantified by LDH release assay after 4 days formation of EBs derived from ES cells defined in d and e. EBs were treated with or without 1 μM RA for last 2 days. Casp8 KD ES cells or EBs were treated with 1 µg/ml Dox throughout the experiments. g Western blot analysis of Casp8, Ripk1, Ripk3, and Mlkl was performed using Tet-On shGFP and Tet-On shCasp8 ES cells after 0–6 days of formation of EBs in the presence or absence of 1 μg/ml Dox. EBs were cultured with or without 1 μM RA after 3 days of formation of EBs. Actin was detected as a control. Molecular weight markers are indicated (kDa). h qRT-PCR analysis of Ripk1, Ripk3, and Mlkl was performed using ES cells defined in g before and after 6 days of EBs formation. i Cell death was quantified by LDH release assay after 4 days of formation of EBs described in c. EBs were treated with or without 1 μM RA for last 2 days in the presence or absence of 10 ng/ml TNFα or 10 ng/ml IFNγ. **p < 0.01 and *p < 0.05.
Fig. 5
Fig. 5
Knockdown of Casp8 expression induced nuclear translocation of RIPK3 to enhance RA signaling. a P19 cells expressing shRipk3 and shRipk3-resistant 3xFlag-Ripk3 were treated with or without 2 ng/ml LMB for 24 h. Subsequently, subcellular localization of 3xFlag-RIPK3 was analyzed by confocal fluorescence microscopy after staining with Alexa Fluor® 488-anti-Flag antibody and DAPI. Scale bars, 20 μm. b P19 cells defined in a and expressing shLacZ were subjected to qRT-PCR analysis of RA-specific target genes, Crabp2, Hoxb1, and Cyp26a1, after treatment with or without 1 μM RA and 2 ng/ml LMB for 24 h. Vector, an empty vector. c Tet-On shCasp8 P19 cells and Tet-On shLacZ P19 cells expressing shRipk3 and shRipk3-resistant 3xFlag-Wt Ripk3 were cultured with or without 1 μg/ml Dox for 4 days. Subsequently, subcellular localization of 3xFlag-RIPK3 was analyzed by confocal fluorescence microscopy after staining with Alexa Fluor® 488-anti-Flag antibody and DAPI. Scale bars, 20 μm. d Western blot analysis of endogenous RIPK3 from nuclear fractions and total cell lysates was carried out using Tet-On shCasp8 ES cells cultured with or without 1 μg/ml Dox for 5 days. Cells were treated with or without 1 μM RA for last 24 h. Cytoplasmic eEF1A1 and nuclear Histone H3 were simultaneously analyzed. Molecular weight markers are indicated (kDa). **p < 0.01 and *p < 0.05
Fig. 6
Fig. 6
RIPK1 and RIPK3 interacted with RARα. P19 cells were transfected with expression vectors encoding EGFP-RIPK1 and/or mCherry-RARα (a), or mCherry-RIPK3 and/or EGFP-RARα (b). Cells were cultured for 48 h, and subcellular localization of these proteins was analyzed by confocal fluorescence microscopy after staining with DAPI. Scale bars, 20 μm. c P19 cells expressing shRipk3 and shRipk3-resistant 3xFlag-Ripk3 were transfected with an expression vector encoding mCherry-RARα or mCherry, and cultured for 48 h. Then, subcellular localization of mCherry-RARα or mCherry was analyzed by fluorescence microscopy after staining with DAPI. Scale bar, 20 µm. d Lysates of HEK293T cells transiently expressing 3xFlag-tagged RIPK3 and/or EGFP-RARα were subjected to immunoprecipitation (IP) with anti-Flag antibody (Flag) or anti-GFP antibody (GFP), and analyzed by western blotting with anti-Flag antibody or anti-GFP antibody. Total cell lysates (input) were also analyzed. Molecular weight markers are indicated (kDa). Dox (1 μg/ml)-treated or -untreated Tet-On shCasp8 ES cells expressing (e) or not expressing (f) shRipk3 and shRipk3-resistant 3xFlag-Ripk3 were analyzed after 4 days formation of EBs. EBs were treated with or without 1 μM RA for last 2 days. Western blot analysis was carried out for immunoprecipitates with control IgG (IgG) or ant-Flag antibody (Flag) (d), and with control IgG or anti-RARα. Total cell lysates (Input) were also analyzed. Hash indicates nonspecific bands and double hashindicates IgG-derived bands
Fig. 7
Fig. 7
RA treatment notably enhanced binding of not only RARs but also RIPK1 and RIPK3 to RAREs of RA-inducible genes in the absence of Casp8 expression. a Tet-On shCasp8 ES cells were cultured with or without 1 μg/ml Dox for 4 days and then treated with or without 1 μM RA for 24 h in the presence or absence of Dox. Subsequently, ChIP analysis for the Rarb-specific RARE or Gapdh promoter region using an anti-RIPK1 antibody was carried out. Tet-On shCasp8 P19 cells expressing shRipk3 (shRipk3 + ) or shLacZ (shRipk3-) together with or without the expression of shRipk3-resistant 3xFlag-Wt RIPK3, kinase-negative 551 A mutant RIPK3, or RHIM AAAA mutant RIPK3 were treated with or without 1 μM RA for 24 h in the presence or absence of 1 μg/ml Dox, and subjected to ChIP analysis for the Cyr26a1-specific RARE or Gapdh promoter region using anti-Flag antibody or control IgG (b), or using anti-RAR antibody or control IgG (c). Vector, an empty vector. d Dox (1 μg/ml)-treated Tet-On shCasp8 ES cells expressing shRipk3 and shRipk3-resistant 3xFlag-Wt RIPK3 were subjected to immunoprecipitation (IP) after 4 days formation of EBs. EBs were treated with 1 μM RA for last 2 days. Immunoprecipitates with control IgG and ant-Flag antibodies were subjected to western blot analysis using indicated antibodies. Molecular weight markers are indicated (kDa). **p < 0.01 and *p < 0.05
Fig. 8
Fig. 8
RA signaling was enhanced in Casp8−/− mouse embryos. a Immortalized MEFs derived from Wt (Casp8+/+) and Casp8−/− embryos were subjected to western blot analysis for Caspase-8 and RIPK3. b qRT-PCR analysis of RA-induced genes, Crabp2, Cyp26a1, and Rarb, was carried out in Casp8+/+ and Casp8−/− MEFs after treatment with or without 1 μM RA for 24 h. c Dual-luciferase reporter analysis of RARE was carried out using RA-treated Casp8+/+ and Casp8−/− MEFs. d qRT-PCR analysis of RA-inducible genes, Crabp2, Cyp26a1, and Rarb, was performed in E10.5 Wt (Casp8+/+) and Casp8−/− littermates (n = 10). RNA was extracted from whole embryos. e Expressions of RA-inducible genes, Crabp2, Cyp26a1, and Rarb were analyzed in E10.5 or E11.5 Wt (Casp8+/+) and Casp8−/− littermates by whole-mount in situ hybridization analysis. Scale bars, 2 mm. f, g Whole-mount in situ hybridization analysis of Rarb and Raldh2 was carried out using E11.5 Casp8−/− embryos (n = 5). Scale bars, 2 mm. Views of heart and AGM (f), and neural tube (g) were shown. Scale bars, 1 mm. h Whole-mount in situ hybridization analysis of Rarb was carried out using representative E11.5 embryos with the indicated genotypes (n = 5). Scale bars, 2 mm. i qRT-PCR analysis of RA-inducible genes, Crabp2, Cyp26a1, and Rarb, was carried out as described in d. **p < 0.01 and *p < 0.05
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