Regulation of death receptor signaling by the autophagy protein TP 53 INP 2
2019; Springer Nature; Volume: 38; Issue: 10 Linguagem: Inglês
10.15252/embj.201899300
ISSN1460-2075
AutoresSaška Ivanova, Mira Polajnar, Álvaro Jesús Narbona‐Pérez, María Isabel Hernández‐Álvarez, Petra Frager, Konstantin Slobodnyuk, Natàlia Plana, Ángel R. Nebreda, Manuel Palacı́n, Roger R. Gomis, Christian Behrends, António Zorzano,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle12 April 2019free access Source DataTransparent process Regulation of death receptor signaling by the autophagy protein TP53INP2 Saška Ivanova Corresponding Author [email protected] orcid.org/0000-0003-0080-6680 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Barcelona, Spain Departament de Bioquimica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Mira Polajnar Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany Munich Cluster for System Neurology, Medical Faculty, Ludwig-Maximilians-University München, Munich, Germany Search for more papers by this author Alvaro Jesus Narbona-Perez Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Maria Isabel Hernandez-Alvarez orcid.org/0000-0003-2483-7000 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Departament de Bioquimica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain Hospital Universitari de Tarragona Joan XXIII, Institut Investigació Sanitaria Pere Virgili (IISPV), Universitat Rovira i Virgili, Tarragona, Spain Search for more papers by this author Petra Frager Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Konstantin Slobodnyuk Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Natalia Plana Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Angel R Nebreda orcid.org/0000-0002-7631-4060 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain ICREA, Insitució Catalana de Recerca i Estudis Avançats, Barcelona, Spain Search for more papers by this author Manuel Palacin Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Barcelona, Spain CIBER de Enfermedades Raras, Barcelona, Spain Search for more papers by this author Roger R Gomis orcid.org/0000-0001-6473-2858 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain ICREA, Insitució Catalana de Recerca i Estudis Avançats, Barcelona, Spain CIBERONC, Barcelona, Spain Departament de Medicina, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Christian Behrends orcid.org/0000-0002-9184-7607 Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany Munich Cluster for System Neurology, Medical Faculty, Ludwig-Maximilians-University München, Munich, Germany Search for more papers by this author Antonio Zorzano Corresponding Author [email protected] orcid.org/0000-0002-1638-0306 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Barcelona, Spain Departament de Bioquimica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Saška Ivanova Corresponding Author [email protected] orcid.org/0000-0003-0080-6680 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Barcelona, Spain Departament de Bioquimica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Mira Polajnar Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany Munich Cluster for System Neurology, Medical Faculty, Ludwig-Maximilians-University München, Munich, Germany Search for more papers by this author Alvaro Jesus Narbona-Perez Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Maria Isabel Hernandez-Alvarez orcid.org/0000-0003-2483-7000 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Departament de Bioquimica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain Hospital Universitari de Tarragona Joan XXIII, Institut Investigació Sanitaria Pere Virgili (IISPV), Universitat Rovira i Virgili, Tarragona, Spain Search for more papers by this author Petra Frager Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Konstantin Slobodnyuk Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Natalia Plana Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Search for more papers by this author Angel R Nebreda orcid.org/0000-0002-7631-4060 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain ICREA, Insitució Catalana de Recerca i Estudis Avançats, Barcelona, Spain Search for more papers by this author Manuel Palacin Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Barcelona, Spain CIBER de Enfermedades Raras, Barcelona, Spain Search for more papers by this author Roger R Gomis orcid.org/0000-0001-6473-2858 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain ICREA, Insitució Catalana de Recerca i Estudis Avançats, Barcelona, Spain CIBERONC, Barcelona, Spain Departament de Medicina, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Christian Behrends orcid.org/0000-0002-9184-7607 Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany Munich Cluster for System Neurology, Medical Faculty, Ludwig-Maximilians-University München, Munich, Germany Search for more papers by this author Antonio Zorzano Corresponding Author [email protected] orcid.org/0000-0002-1638-0306 Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Barcelona, Spain Departament de Bioquimica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain Search for more papers by this author Author Information Saška Ivanova *,1,2,3, Mira Polajnar4,5,6, Alvaro Jesus Narbona-Perez1, Maria Isabel Hernandez-Alvarez1,3,7, Petra Frager1, Konstantin Slobodnyuk1, Natalia Plana1, Angel R Nebreda1,8, Manuel Palacin1,2,9, Roger R Gomis1,8,10,11, Christian Behrends4,6 and Antonio Zorzano *,1,2,3 1Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain 2CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Barcelona, Spain 3Departament de Bioquimica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain 4Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany 5German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany 6Munich Cluster for System Neurology, Medical Faculty, Ludwig-Maximilians-University München, Munich, Germany 7Hospital Universitari de Tarragona Joan XXIII, Institut Investigació Sanitaria Pere Virgili (IISPV), Universitat Rovira i Virgili, Tarragona, Spain 8ICREA, Insitució Catalana de Recerca i Estudis Avançats, Barcelona, Spain 9CIBER de Enfermedades Raras, Barcelona, Spain 10CIBERONC, Barcelona, Spain 11Departament de Medicina, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain *Corresponding author. Tel: +34934034701; E-mail: [email protected] *Corresponding author. Tel: +34934037197; E-mail: [email protected] EMBO J (2019)38:e99300https://doi.org/10.15252/embj.201899300 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract TP53INP2 positively regulates autophagy by binding to Atg8 proteins. Here, we uncover a novel role of TP53INP2 in death-receptor signaling. TP53INP2 sensitizes cells to apoptosis induced by death receptor ligands. In keeping with this, TP53INP2 deficiency in cultured cells or mouse livers protects against death receptor-induced apoptosis. TP53INP2 binds caspase-8 and the ubiquitin ligase TRAF6, thereby promoting the ubiquitination and activation of caspase-8 by TRAF6. We have defined a TRAF6-interacting motif (TIM) and a ubiquitin-interacting motif in TP53INP2, enabling it to function as a scaffold bridging already ubiquitinated caspase-8 to TRAF6 for further polyubiquitination of caspase-8. Mutations of key TIM residues in TP53INP2 abrogate its interaction with TRAF6 and caspase-8, and subsequently reduce levels of death receptor-induced apoptosis. A screen of cancer cell lines showed that those with higher protein levels of TP53INP2 are more prone to TRAIL-induced apoptosis, making TP53INP2 a potential predictive marker of cancer cell responsiveness to TRAIL treatment. These findings uncover a novel mechanism for the regulation of caspase-8 ubiquitination and reveal TP53INP2 as an important regulator of the death receptor pathway. Synopsis Death cell receptors initiate apoptosis, necroptosis or inflammation depending on their cell-surface expression, ligand concentration and intracellular context. The autophagy regulator TP53INP2 promotes death receptor-induced apoptosis through TRAF6-dependent ubiquitination of caspase-8. TP53INP2 sensitizes cancer cells to death receptor-induced apoptosis. Knockout of TP53INP2 protects cultured cells and mouse livers from FasL-induced apoptosis. TP53INP2 binds TRAF6 E3 ligase and ubiquitinated caspase-8 via its TRAF6- and ubiquitin-interacting motifs, respectively, to enhance caspase-8 poly-ubiquitination. High TP53INP2 levels may predict responsiveness of cancer cells to TRAIL treatment. Introduction Apoptosis is a process of programmed cell death that is crucial for the homeostasis of an organism, and its deregulation occurs in several pathologies (Jacobson et al, 1999; Vaux & Korsmeyer, 1999). Apoptosis can be triggered through either an intrinsic or extrinsic pathway (Ferri & Kroemer, 2001; Fulda & Debatin, 2006). In the former, cellular damage is sensed by various Bcl-2 pro-apoptotic homologues and leads to Bax/Bak oligomerization in the outer mitochondrial membrane, release of cytochrome c, and apoptosome formation, where caspase-9 is activated (Zou et al, 1999). Activated caspase-9 cleaves caspases-3, -6, and -7, which execute apoptosis (Zou et al, 1999). In the extrinsic pathway, death ligands (FasL, TRAIL/Apo2L and TNFα) bind to their cognitive receptors and induce their trimerization, thereby allowing subsequent binding of the adaptor protein FADD and caspase-8 to the DISC complex (death-inducing signaling complex; Medema et al, 1997). Caspase-8 was thought to be activated through the so-called proximity-induced model, i.e., dimerization of pro-caspase-8 molecules in the DISC complex. However, this model has recently been challenged by the DED chain assembly model, which proposes that a FADD molecule interacts with several caspase-8 molecules (Dickens et al, 2012; Schleich et al, 2012). Activated caspase-8 directly cleaves executioner caspases (i.e., caspase-3) in type I cells (e.g., thymocytes), while in type II cells (e.g., hepatocytes) it cleaves the Bcl-2 homology domain 3 (BH3) protein Bid, producing tBid, which amplifies the signal through mitochondria (Li et al, 1998; Luo et al, 1998). Recently, several studies have added additional layers of complexity to caspase-8 activation, revealing that ubiquitination plays a key role in this process. For example, the E3 ubiquitin ligase cullin-3 ubiquitinates caspase-8 at its C-terminus in the DISC complex and ubiquitinated caspase-8 is aggregated by p62 for its full activation (Jin et al, 2009). In contrast, TRAF2 adds K48-ubiquitin chains to the large catalytic domain of caspase-8 and marks it for degradation (Gonzalvez et al, 2012), while HECTD3 ubiquitination of the Lys residue between the DED and the large domain of caspase-8 increases the threshold for death receptor-induced apoptosis (Li et al, 2013). One of the hallmarks of cancer cells is their capacity to evade apoptosis. Most chemotherapies in clinical practice aim to induce cell death in tumors, thus shrinking the tumors to a size that can be removed by surgery, or to kill any remaining and/or circulating tumor cells. Chemotherapy has several disadvantages. In addition to not being effective in all or even in the majority of patients, it causes side effects. This observation points to the need for personalized medicine, i.e., the selection of patients that will respond and benefit from a given chemotherapy. The initial optimism caused by the discovery of TRAIL, for example, which selectively kills cancer cells, plummeted after several unsuccessful clinical trials. Thus, better antagonists and molecular markers to identify patients who would respond to TRAIL are needed (de Miguel et al, 2016; von Karstedt et al, 2017). This need is further emphasized by the observation that tumors not undergoing apoptosis upon TRAIL administration can diverge the signaling to cytokine production, thus favoring tumor growth (Hartwig et al, 2017; Henry & Martin, 2017). The complexity of the cross-talk between autophagy and apoptosis has been widely studied, not only with the purpose of understanding the underlying mechanisms, but also of modulating both pathways in tumors. Several autophagic proteins have a dual role in both processes (Yousefi et al, 2006; Cho et al, 2009; Giansanti et al, 2011). The inhibition of autophagy causes the accumulation of autophagosomal membranes, which serve as platforms for intracellular DISC formation (Laussmann et al, 2011; Pan et al, 2011; Young et al, 2012; Huang et al, 2013). Canonical and intracellular DISC formation occurs independently and requires distinct membranes (Jiang et al, 2011; Laussmann et al, 2011; Pan et al, 2011; Young et al, 2012; Deegan et al, 2014). Thus, pro-caspase-8 binds to intracellular DISC on the phagophore through ATG12-ATG5-FADD on the outer membrane or through LC3-p62 on the inner membrane of the accumulating autophagosomes (Bell et al, 2008; Jiang et al, 2011; Laussmann et al, 2011; Pan et al, 2011; Young et al, 2012; Huang et al, 2013; Deegan et al, 2014; Tang et al, 2017). The LC3-p62 axis most probably recruits ubiquitinated caspase-8 in a similar way as the autophagic cargo is recruited to autophagosomes (Pankiv et al, 2007; Huang et al, 2013). In proliferating cells, TP53INP2 is a nuclear protein that interacts with nuclear hormone receptors (Baumgartner et al, 2007; Francis et al, 2010), shuttles from the nucleus to the cytosol (Mauvezin et al, 2010, 2012), and stimulates protein synthesis by promoting ribosomal biogenesis in the nucleolus (Xu et al, 2016). However, upon nutrient depletion, TP53INP2 interacts with a nuclear and deacetylated pool of LC3 and shuttles it rapidly to the cytosol to initiate autophagy (Huang et al, 2015). TP53INP2 is a positive regulator of autophagy, and it interacts directly with the LIR sequence of all Atg8 family members (Nowak et al, 2009; Mauvezin et al, 2010; Sancho et al, 2012). We recently showed that TP53INP2 is also an ubiquitin-binding protein, with a preference for mono- and K63-linked ubiquitin chains (Sala et al, 2014). Here, we identified an unexpected role of TP53INP2 in death receptor signaling. We show that TP53INP2 sensitizes various cancer cell lines to death receptor-induced apoptosis. We observed that TP53INP2 increases the activation of caspase-8 by upregulating its K63-ubiquitination levels in a TRAF6-dependent manner. Furthermore, we demonstrate that TP53INP2 acts as a scaffold for caspase-8 polyubiquitination by TRAF6. In addition, we show that cancer cell lines with high protein levels of TP53INP2 respond better to TRAIL-induced apoptosis than those with no or low amounts of TP53INP2. This observation indicates that TP53INP2 might be a potential biomarker for personalized TRAIL treatment in cancers where caspase-8 protease activity is intact. Altogether, our findings demonstrate that TP53INP2 acts as a switch at the level of caspase-8 activation, favoring death receptor-mediated apoptosis. Results TP53INP2 regulates death receptor-induced apoptosis Several autophagic proteins participate in the cross-talk between autophagy and apoptosis (Yousefi et al, 2006; Cho et al, 2009; Laussmann et al, 2011; Huang et al, 2013; Strappazzon et al, 2016). In this regard, we examined the role of TP53INP2 in apoptosis. We overexpressed TP53INP2 in HeLa cells, which express undetectable levels of this protein, and we induced cell death by various agents. Surprisingly, TP53INP2 increased the sensitivity of cells to death induced by ligands of death receptors more efficiently than to other inducers (Fig 1A and Appendix Fig S1A). This observation prompted us to explore the role of TP53INP2 in death receptor-induced cell death. Using inhibitors of apoptosis (zVAD) and necrosis (necrostatin-1/Nec-1), annexin V staining [flow cytometry measurement of phosphatidylserine exposure (PS)] and DEVDase activity (indicative of caspase activity), we confirmed that TP53INP2 sensitizes cells to death receptor-induced apoptosis and not necroptosis (Fig 1B and C, and Appendix Fig S1B). Furthermore, the activation of caspase-8 and caspase-3, and detection of the caspase-generated 85 kDa fragment of PARP-1 were higher in cells expressing TP53INP2 and treated with a range of concentrations of FasL and TRAIL (Fig 1D and E). TNFα induces apoptosis by activation of the death receptor pathway by a process that is stimulated to cycloheximide (Kreuz et al, 2001), and TNFα-induced apoptosis was further augmented by TP53INP2 overexpression in HeLa cells (Appendix Fig S1C and D). Similar results were obtained in MDA231 and MCF7 cells treated with FasL (Appendix Fig S1E and F). LC3-II protein levels were increased in TP53INP2-overexpressing cells as previously described (Fig 1D and E; Sala et al, 2014). Moreover, time-course experiments showed that FasL-induced apoptosis in TP53INP2-expressing cells occurs faster than in control cells. In the former, apoptosis started around 4 h post-induction (Fig 1F). At this time point, caspase-3 cleavage was detected and the levels of TP53INP2 were the highest (Fig 1F), thereby supporting the notion that TP53INP2 accumulates in the first 4 h of FasL treatment and that TP53INP2 contributes to faster activation of apoptosis triggered by death receptors. Similar results were obtained by time-course experiment with TRAIL (Fig 1G). Since TP53INP2 is cleaved during apoptosis and we detected a cleavage product of approximately 26 kDa (Fig 1D–G), we explored whether the sensitization of TP53INP2 to death receptor-induced apoptosis involves its cleavage by caspases. To test this, we first mutated aspartate residues at potential caspase cleavage sites (Fig 1H) to glutamate in order to produce a caspase-noncleavable mutant form (TP53INP2 3DE). Indeed, recombinant caspase-3 cleaved the wild-type TP53INP2 but not the 3DE mutant (Appendix Fig S1G); however, the 3DE mutant did not abolish the sensitization effect of TP53INP2 to FasL-induced apoptosis (Fig 1I and J). This observation indicates that the mechanism involved does not require the cleavage of TP53INP2 by caspases. Figure 1. TP53INP2 sensitizes cells to death receptor-induced apoptosis A. HeLa cells were treated with the indicated inducers of apoptosis for 24 h, and cell viability was assessed by annexin V and PI staining. B. The percentage of viable cells 24 h after FasL (50 ng/ml) and TRAIL (10 ng/ml) treatment in the absence or presence of z-VAD-fmk (20 μM) or Nec-1 (30 μM). C. Quantification of DEVDase activity in HeLa cells after 16 h of FasL or 4 h of TRAIL treatment. D, E. HeLa cells were infected with adenovirus for LacZ or TP53INP2 and treated with different concentrations of FasL for 16 h (D) or TRAIL for 4 h (E). Cell lysates were then subjected to Western blot analysis for various apoptotic and autophagic markers. F. Time-dependent cleavage of PARP-1, caspase-8, caspase-3, and TP53INP2 during FasL-induced apoptosis (50 ng/ml) in HeLa cells expressing LacZ or TP53INP2. G. Time-dependent cleavage of PARP-1, caspase-8, caspase-3, and TP53INP2 during TRAIL-induced apoptosis (50 ng/ml) in HeLa cells expressing LacZ or TP53INP2. H. Schematic presentation of caspase cleavage sites in human and mouse TP53INP2. LIR; LC3 interacting region. I. DEVDase activity quantification in lysates of HeLa cells expressing LacZ, wt TP53INP2 or TP53INP2 3DE mutant in control or FasL-treated cells (50 ng/ml; 16 h). J. HeLa cells were transduced with adenovirus for LacZ, wt TP53INP2 or 3DE mutant, and cell lysates were subjected to Western blot analysis with indicated antibodies. Data information: Data are given as mean ± SEM and were analyzed by two-way Student's t-test; n = three independent experiments, *P < 0.05. Source data are available online for this figure. Source Data for Figure 1 [embj201899300-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint In contrast, HeLa cells depleted of TP53INP2 by CRISPR technology were less sensitive to treatment with FasL or TRAIL (Fig 2A–H). Thus, percentage of cell death was lower in TP53INP2-deficient cells compared to wild-type cells upon FasL or TRAIL (Fig 2B), and similarly, cleaved caspase-3 or PARP-1 was reduced in TP53INP2-deficient cells treated with FasL or TRAIL (Fig 2C–H). Figure 2. Loss of TP53INP2 renders cells resistant to death receptor-induced apoptosis A. Western blot analysis of TP53INP2 in wild-type (wt) HeLa and TP53INP2 CRISPR KD cells. B. Cell viability of HeLa wt and TP53INP2 KD cells after 24 h treatment with FasL (50 ng/ml) or TRAIL (10 ng/ml) measured as annexin V- and PI-negative cells. C. HeLa control cells and TP53INP2 CRISPR KD cells were treated with different concentrations of FasL for 16 h, and cell lysates were subjected to Western blot analysis for PARP and caspase-3 cleavage. D, E. Quantification of protein levels of cleaved PARP (D) and cleaved caspase-3 (E) after FasL treatment. F. HeLa control cells and TP53INP2 CRISPR KD cells were treated with different concentrations of TRAIL for 4 h, and cell lysates were subjected to Western blot analysis for PARP1, caspase-3, and caspase-8 cleavage. G, H. Quantification of protein levels of cleaved PARP (G) and cleaved caspase-3 (H) after TRAIL treatment. I. Detection of TP53INP2 protein levels in livers of control and TP53INP2 L-KO mice. J. Control and TP53INP2 KO hepatocytes were treated with FasL for 16 h, and levels of cleaved caspase-3 were detected by Western blot. K. Quantification of protein levels of cleaved caspase-3 after FasL treatment. Data information: Data are given as mean ± SEM and were analyzed by two-way Student's t-test; n = three independent experiments, *P < 0.05. Source data are available online for this figure. Source Data for Figure 2 [embj201899300-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Furthermore, we generated liver-specific TP53INP2 KO mice (L-KO) by crossing Tp53inp2loxP/loxP mice (Sala et al, 2014) with mice expressing Cre recombinase under the control of the albumin promoter. TP53INP2 protein levels were undetectable in livers of L-KO animals, and no changes in the protein levels in other tissues were detected (Fig 2I; Appendix Fig S2A and B). L-KO male and female mice showed normal body weight or blood glucose levels (Appendix Fig S2C and D). Primary hepatocytes isolated from these mice were less susceptible to FasL- and TNFα-induced apoptosis than hepatocytes from control mice (Fig 2J and K, and Appendix Fig S2E). Introducing TP53INP2 back to the L-KO hepatocytes by adenoviral infection restored the capacity of FasL or TNFα to induce apoptosis, as reflected by increased amounts of cleaved PARP-1 and cleaved caspase-3 (Appendix Fig S2E). Of note, overexpression of TP53INP2 per se in control and L-KO hepatocytes was pro-apoptotic (Appendix Fig S2E), thus making high amounts of TP53INP2 toxic for the liver. Taken together, our results show that TP53INP2 increases susceptibility to death receptor-induced apoptosis and that it does so upstream of caspase activation, i.e., before TP53INP2 is cleaved by caspases. Loss-of-function of TP53INP2 protects livers from FasL-induced apoptosis in vivo We next tested whether TP53INP2 regulates death receptor-induced apoptosis in vivo. Control and L-KO mice were injected intraperitoneally with PBS or FasL for 4 h. The cleavage product of PARP-1 and cleaved caspase-3 were detected in controls, but were absent or present to a lesser extent in L-KO mice treated with FasL (Fig 3A and B). Of note, protein levels of p62 were increased and LC3II decreased in L-KO mice treated with PBS (Fig 3A), which is in keeping with prior observations in TP53INP2-deficient skeletal muscle (Sala et al, 2014). In addition, immunohistochemical staining showed more cleaved caspase-3 positive cells in control livers treated with FasL than in L-KO livers (Fig 3C and D). Analysis of TUNEL-positive cells further confirmed more apoptotic cells in control livers treated with FasL than in L-KO livers (Fig 3E and F). Given that caspase-8 activation is not sufficient to directly cleave caspase-3 in liver cells and the signal is amplified through mitochondria, we also checked by immunofluorescence the release of cytochrome c from mitochondria. TOM20 was used as marker of mitochondria. As expected, cytochrome c colocalized with TOM20 in PBS-treated livers of control and L-KO mice (Fig 3G and H; and Appendix Fig S3A). However, in FasL-treated livers of control animals, cytochrome c did not completely colocalize with this marker, indicating the release of cytochrome c from mitochondria (Fig 3G and H; and Appendix Fig S3A) and apoptosome formation, leading to the activation of executioner caspases (i.e., caspase-3). In contrast, colocalization of TOM20 and cytochrome c was detected in FasL-treated L-KO mice (Fig 3G and H, and Appendix Fig S3A). Figure 3. TP53INP2 deficiency protects livers from FasL-induced apoptosis Control and TP53INP2 L-KO animals were injected with PBS or FasL i.p. for 4 h. Whole liver lysates were subjected to immunodetection of the indicated apoptotic and autophagic markers. Quantification of Western blot of cleaved caspase-3 and PARP. Data are presented as mean ± SEM of four samples. Cleaved caspase-3 immunohistochemistry staining of livers from control and TP53INP2 L-KO mice treated with PBS or FasL (Scale bar, 100 μm). Quantification of cleaved caspase-3 immunohistochemistry staining. Data are presented as mean ± SEM of nine different fields (per mice) where in each field more than 200 cells were counted. TUNEL immunohistochemistry staining of livers from control and TP53INP2 L-KO mice treated with PBS or FasL (Scale bar, 100 μm). Quantification of TUNEL immunohistochemistry staining. Data are presented as mean ± SEM of more than 1,500 cells counted per mice (four mice in each experimental group). TOM20 (red) and cytochrome C (green) immunohistofluorescence staining of livers from control and TP53INP2 L-KO mice (Scale bar, 25 μm). Quantification of cells with disperse cytochrome c staining. Data are presented as mean ± SEM of more than 300 cells counted per each experimental group. Data information: Two-way Student's t-test was performed, *P < 0.05. Source data are available online for this figure. Source Data for Figure 3 [embj201899300-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint To determine whether TP53INP2 deficiency has an impact on death receptor independent pathway, control and KD HeLa cells were treated with agents that induce intrinsic cell death such as doxorubicin, cisplatin, or actinomycin D. These agents reduced cell viability in a time-dependent manner (Fig 4A). However, only upon actinomycin D treatment, a significant increase in viability was detected in TP53INP2-deficient cells (Fig 4A). DEVD
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