Casp8 Activity Research Articles

Intrinsic and Extrinsic Apoptotic Pathways May Dictate In Vitro Fertilization Outcomes in Women of Advanced Maternal Age [ID 2683425

INTRODUCTION: Women of advanced maternal age (AMA) (≥35 years) are at an increased risk for a decline in ovarian reserve and have a decreased rate of live birth after one cycle of in vitro fertilization (IVF). Current practice recommends preimplantation genetic testing for aneuploidy (PGT-A) to women of AMA to increase live birth outcomes. However, recent studies have found that conventional IVF was non-inferior to PGT-A. Identifying additional biomarkers may provide another metric for selecting the most viable embryo. METHODS: Blastocoel fluid-conditioned media was collected from day 5 IVF embryos that underwent PGT-A. RNA was purified from pooled blastocoel fluid samples from four groups based on implantation status and maternal age, and then cDNA was synthesized. RT-qPCR was used to assess expression of CASP3, CASP7, CASP8, and BCL2L12 levels in a total of 64 embryos. RESULTS: CASP3 and CASP7 expression were higher in the media from IVF embryos with positive implantation outcomes from patients aged less than 35 years. There was increased CASP8 expression in media from IVF embryos from AMA patients regardless of implantation status. CONCLUSION: Preliminary analysis suggests increased CASP8 expression is associated with successful implantation outcomes in AMA patients. This suggests that CASP8 could be activated in preimplantation embryos as an aging response as found in AMA media samples. One explanation is that CASP8 is associated with aneuploidy in the AMA samples. Further research will determine whether expression and activation of CASP8 are required for positive implantation outcomes and be used as an additional embryo selection tool for other AMA patients utilizing IVF.

The Transcription Factor PU.1 Controls Alternative Splicing of Cflip By Binding to SRSF2/10 Splice Factors in AML Therapy Responses

cFLIP (CFLAR) is an inactive homolog of caspase 8 that functions downstream of death-receptor mediated apoptosis. CFLAR gene is alternatively spliced and exists in three isoforms: a long isoform (cFLIPL), which partially inhibits CASP8 activity, and two short forms (cFLIPR and cFLIPS) that entirely block caspase 8 activity. Moreover, cFLIPL is crucial for macrophage differentiation. Therefore, the ratio between cFLIPL and cFLIPS/R may regulate myeloid differentiation. The transcription factor PU.1 (SPI1) is necessary for myeloid differentiation as well as myeloid cell survival. PU.1 is expressed at low levels in hematopoietic progenitors and increasing levels of PU.1 promote macrophage and granulocyte differentiation. Additionally, aberrant low PU.1 expression contributes to an immature myeloid phenotype, e.g. seen in acute myeloid leukemia (AML). Interestingly, two studies indicated that high PU.1 protein levels were associated with alternative splicing in a murine erythroleukemia model by either direct binding to splice factors or RNA. Based on these observations we hypothesized that PU.1 controls alternative splicing of cFLIP during myeloid differentiation. In line with above hypothesis, differentiating primary CD34+ hematopoietic progenitors into granulocytes and monocytes or AML cells into granulocytes shifted cFLIP splicing in favor of cFLIPL paralleled by increased PU.1 mRNA and protein levels. Next, we assessed the direct impact of PU.1 protein on splicing during granulocytic differentiation by knocking down PU.1 in NB4 acute promyelocytic leukemia (APL) and MOLM13 AML cells. Silencing PU.1 in NB4 and MOLM13 cells decreased cell death and differentiation during all-trans retinoic acid (ATRA)-mediated granulocytic differentiation. Moreover, knocking down PU.1 reduced cFLIPL/S protein and mRNA ratios during ATRA treatment. Interestingly, increasing the cFLIPL/S ratio by knocking down specifically cFLIPS or overexpressing cFLIPL reduced MOLM13 cell viability during ATRA treatment. Since the canonical function of CFLAR isoforms is the inhibition of extrinsic apoptosis, we next tested if increased cFLIPS levels found in AML cells depleted for PU.1 would show reduced sensitivity to TRAIL treatment. Indeed, TRAIL-induced extrinsic apoptosis via TRAIL receptor was significantly reduced in PU.1 depleted MOLM-13 AML cells. To investigate if PU.1 is directly involved in CFLAR splicing, we analyzed the CFLAR pre-mRNA sequence encompassing the alternative splicing site for PU.1 and splice factor binding motifs. We identified one putative PU.1 and SRSF2 as well as SRSF10 splice factor binding sites in the CFLAR pre-mRNA sequence. Using RNA immunoprecipitation, PU.1 pull-down and proximity ligation assays, we showed that all three proteins bind the CFLAR mRNA and physically interact with each other. Lastly, we asked if PU.1 additionally influences cFLIP alternative splicing via regulating splice factor transcription. Knocking down PU.1 in MOLM13 cells resulted in significantly reduced SRSF2 and SRSF10 mRNA and protein levels. We confirmed PU.1 binding to their promoter using ChIP assays. Accordingly, these splice factors showed altered mRNA expression when comparing mature granulocytes to primary AML cells. Since silencing SRSF2 and SRSF10 switched the CFLARL/S isoform ratio towards the long isoform, we suggest that PU.1-mediated induction of these splice factores represents a negative feedback loop. In conclusion, we provide evidence that PU.1 directly regulates alternative splicing of CFLAR during ATRA-mediated neutrophil differentiation and TRAIL-induced cell death in human AML cells. We propose a model where PU.1 binds to CFLAR pre-mRNA and blocks SRSF2 and SRSF10-mediated splicing ultimatively leading to exon 7 skipping and predominant expression of cFLIPL.

Cardiolipin in Immune Signaling and Cell Death.

Cardiolipin (CL) is a tetra-acylated diphosphatidylglycerol lipid. In physiological conditions, CL presents unsaturated chains and is located in the inner mitochondria membrane (IMM). Dead signals, infection, or disease may change the level of CL saturation and oxidation and cause its translocation to the cytosolic side of the outer mitochondrial membrane (OMM), affecting mitochondrial function and the inflammatory response. In this review, we summarize the emerging proapoptotic, pro-, and anti-inflammatory functions of cytosolic-exposed CL and how they are regulated by CL chain saturation and oxidation. We underline how the unique dimeric phospholipid structure confers peculiar properties on CL in the regulation of cell death and immune system proteins, such as the Nucleotide-binding domain and leucine-rich repeat-containing pyrin protein 3 (NLRP3), caspases (Casp), and Toll-like receptor 4 (TLR4). We also provide an overview of the human diseases in which CL deficiency or modification are implicated and of the use of exogenous unsaturated CL (uCL) as a novel therapeutic approach.

Gsta4 controls apoptosis of differentiating adult oligodendrocytes during homeostasis and remyelination via the mitochondria-associated Fas-Casp8-Bid-axis

Arrest of oligodendrocyte (OL) differentiation and remyelination following myelin damage in multiple sclerosis (MS) is associated with neurodegeneration and clinical worsening. We show that Glutathione S-transferase 4α (Gsta4) is highly expressed during adult OL differentiation and that Gsta4 loss impairs differentiation into myelinating OLs in vitro. In addition, we identify Gsta4 as a target of both dimethyl fumarate, an existing MS therapy, and clemastine fumarate, a candidate remyelinating agent in MS. Overexpression of Gsta4 reduces expression of Fas and activity of the mitochondria-associated Casp8-Bid-axis in adult oligodendrocyte precursor cells, leading to improved OL survival during differentiation. The Gsta4 effect on apoptosis during adult OL differentiation was corroborated in vivo in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis models, where Casp8 activity was reduced in Gsta4-overexpressing OLs. Our results identify Gsta4 as an intrinsic regulator of OL differentiation, survival and remyelination, as well as a potential target for future reparative MS therapies.

COP9 Signalosome Suppresses RIPK1-RIPK3-Mediated Cardiomyocyte Necroptosis in Mice.

Mechanisms governing the induction of heart failure by the impairment of autophagy and the ubiquitin-proteasome system and the molecular pathways to cardiomyocyte necrosis remain incompletely understood. COPS8 is an essential subunit of the COP9 (COnstitutive Photomorphogenesis 9) signalosome, a key regulator of ubiquitination. Mice with cardiomyocyte-restricted knockout of Cops8 (Cops8-cko) show autophagic and ubiquitin-proteasome system malfunction and massive cardiomyocyte necrosis followed by acute heart failure and premature death, providing an excellent animal model to address the mechanistic gaps specified above. This study was conducted to determine the nature and underlying mechanisms of the cardiomyocyte necrosis in Cops8-cko mice. Compared with littermate control mice, myocardial protein levels of key factors in the necroptotic pathway (RIPK1 [receptor-interacting protein kinase 1], RIPK3, MLKL [mixed lineage kinase-like], the RIPK1-bound RIPK3), protein carbonyls, full-length Casp8 (caspase 8), and BCL2, as well as histochemical staining of superoxide anions were significantly higher but the cleaved Casp8 and the Casp8 activity were significantly lower in Cops8-cko mice. In vivo cardiomyocyte uptake of Evan's blue dye was used as an indicator of necrosis. Cops8-cko mice treated with a RIPK1 kinase inhibitor (Nec-1 [Necrostatin-1]) showed less Evans blue dye uptake (0.005% versus 0.20%; P<0.0001) and longer median lifespan (32.5 versus 27 days; P<0.01) than those treated with vehicle control. RIPK3 haploinsufficiency showed similar rescuing effects on Cops8-cko but Cyclophilin D deficiency did the opposite. Cardiac Cops8/COP9 signalosome malfunction causes RIPK1-RIPK3 dependent, but mitochondrial permeability transition pore independent, cardiomyocyte necroptosis in mice and the COP9 signalosome plays an indispensable role in suppressing cardiomyocyte necroptosis.

Glucocorticoid modulatory element-binding protein 1 (GMEB1) interacts with the de-ubiquitinase USP40 to stabilize CFLARL and inhibit apoptosis in human non-small cell lung cancer cells

BackgroundGMEB1 was originally identified via its interaction with GMEB2, which binds to the promoter region of the tyrosine aminotransferase (TAT) gene and modulates transactivation of the glucocorticoid receptor gene. In the cytosol, GMEB1 interacts with and inhibits CASP8, but the molecular mechanism is currently unknown.MethodsHuman non-small cell lung cancer cells and 293FT cells were used to investigate the function of GMEB1/USP40/CFLARL complex by WB, GST Pull-Down Assay, Immunoprecipitation, Immunofluorescence and Flow cytometry analysis. A549 cells overexpressing green fluorescent protein and GMEB1 shRNA were used for tumor xenograft using female athymic nu/nu 4-week-old mice.ResultsWe found GMEB1 interacted with CFLARL (also known as c-FLIPL) in the cytosol and promoted its stability. USP40 targeted CFLARL for K48-linked de-ubiquitination. GMEB1 promoted the binding of USP40 to CFLARL. USP40 knockdown did not increase CFLARL protein level despite GMEB1 overexpression, suggesting GMEB1 promotes CFLARL stability via USP40. Additionally, GMEB1 inhibited the activation of pro-caspase 8 and apoptosis in non-small cell lung cancer (NSCLC) cell via CFLARL stabilization. Also, GMEB1 inhibited the formation of DISC upon TRAIL activation. CFLARL enhanced the binding of GMEB1 and CASP8. Downregulation of GMEB1 inhibited A549 xenograft tumor growth in vivo.ConclusionsOur findings show the de-ubiquitinase USP40 regulates the ubiquitination and degradation of CFLARL; and GMEB1 acts as a bridge protein for USP40 and CFLARL. Mechanistically, we found GMEB1 inhibits the activation of CASP8 by modulating ubiquitination and degradation of CFLARL. These findings suggest a novel strategy to induce apoptosis through CFLARL targeting in human NSCLC cells.

Influence of Fat on Differential Receptor Interacting Serine/Threonine Protein Kinase 1 Activity Leading to Apoptotic Cell Death in Murine Liver Ischemia Reperfusion Injury Through Caspase 8.

Current understanding is that receptor interacting serine/threonine protein kinase 1 (RIPK1) can lead to two distinct forms of cell death: RIPK3‐mediated necroptosis or caspase 8 (Casp8)‐mediated apoptosis. Here, we report that RIPK1 signaling is indispensable for protection from hepatocellular injury in a steatotic liver undergoing ischemia reperfusion injury (IRI) but not in the lean liver. In lean liver IRI, RIPK1‐mediated cell death is operational, leading to protection in RIP1 kinase‐dead knock‐in (RIPK1K45A) mice and necrostatin‐1s (Nec1s)‐treated lean wild‐type (WT) mice. However, when fed a high‐fat diet (HFD), RIPK1K45A‐treated and Nec1s‐treated WT mice undergoing IRI demonstrate exacerbated hepatocellular injury along with decreased RIPK1 ubiquitylation. Furthermore, we demonstrate that HFD‐fed RIPK3–/–/Casp8–/– mice show protection from IRI, but HFD‐fed RIPK3–/–/Casp8–/+ mice do not. We also show that blockade of RIPK1 leads to increased Casp8 activity and decreases mitochondrial viability. Conclusion: Although more studies are required, we provide important proof of concept for RIPK1 inhibition leading to distinctive outcomes in lean and steatotic liver undergoing IRI. Considering the rising incidence of nonalcoholic fatty liver disease (NAFLD) in the general population, it will be imperative to address this critical difference when treating patients with RIPK1 inhibitors. This study also presents a new target for drug therapy to prevent hepatocellular injury in NAFLD.

FAS-Mediated Apoptosis Assay in Patients with ALPS-like Phenotype Carrying CASP10 Mutations

INTRODUCTIONAutoimmune lymphoproliferative syndrome (ALPS) is a rare congenital disorder characterized by an impaired FAS-mediated apoptosis that leads to chronic benign lymphoproliferation and autoimmunity. In most cases mutation on FAS gene are responsible of the disease and the phenotype of individuals carrying the same variants can vary from asymptomatic/mild forms to severe disease, due to incomplete penetrance of pathogenic mutations. More rarely, other genes involved in this pathway, such as CASP10, are mutated. Few clinical and molecular data have been reported on small numbers of patients carrying CASP10 mutation showing that different genetic variations can produce contrasting phenotypic effects. So far, 2 mutations have been recognized as pathogenic (I406L and L258F) and other have been reported with controversial result on their pathogenicity (V410l, Y446C) or are known to be polymorphic variants (L522l) AIMSThe aim of this study is to evaluate the FAS-mediated apoptosis function in patients with ALPS or ALPS-like symptoms carrying mutations on CASP10 gene. METHODSWe evaluated FAS-mediated apoptosis pathway in all patients presenting with an ALPS/ALPS-like phenotype that were found to carry a CASP10 mutation in our Institution. Molecular findings were obtained by NGS analysis of a panel of genes involved in the most common immune-dysregulation syndromes and immune-deficiencies and were confirmed by Sanger sequencing. Functional studies were performed by Western blot analysis of CASP10, CASP8, and PARP proteins after TRIAL-induced apoptosis stimulation. Healthy individuals were used as control. RESULTSWe identified 6 patients with ALPS (2) or ALPS-like (4) phenotype, carrying the following heterozygous CASP10 mutations: I406L (1), V410l (2), Y446C (1) L522l (2). Western blot analysis showed an impaired activation of CASP10, CASP8, and PARP proteins in all cases compared to healthy control (Fig. 1) CONCLUSIONSIn our symptomatic patients, the CASP10 polymorphic variant L522l and other mutations whose pathogenicity is controversial (V410l, Y446C) were associated with impaired CASP10, CASP8, PARP activity -and therefore with apoptosis dysfunction- as in the case of I406L pathogenic mutation. We can speculate that, in addition to the functional impairment of apoptosis, other genetic and epigenetic factors might be crucial for the development of clinical symptoms in CASP10 mutated patients, as already described in FAS mutations, suggesting that the search of other mutations in patients with ALPS/ALPS-like phenotype should be encouraged. Nonetheless, further studies on epigenetic factors potentially implicated in the expression of symptoms are needed to fully understand the heterogeneity of clinical phenotype of this disorder. DisclosuresNo relevant conflicts of interest to declare.

A Novel PU.1 - Caspase 8/cFLIP Axis in Neutrophil and Macrophage Differentiation of AML Cells

The transcription factor PU.1 (SPI-1) plays an important role in numerous cellular processes of myeloid cells, such as cell survival, proliferation and differentiation. PU.1 is expressed at intermediate levels in hematopoietic progenitor cells, whereas progenitors expressing low amounts of PU.1 differentiate towards the lymphoid lineage, while increased amounts of PU.1 levels promoted macrophage or granulocyte differentiation. Additionally, low PU.1 expression levels contribute to the immature myeloid phenotype in acute myeloid leukemia (AML).We recently linked PU.1 expression to TRAIL and chemotherapy sensitivity in AML cells. To further molecularly dissect the functions of PU.1 in myeloid cells, we focused on putative PU.1 targets associated with cell death responses emerging from a gene expression profiling experiment using Pu.1-null and Pu.1-restored 503 murine myeloid cells. We identified Caspase 8 (Casp8) and its paralogous gene c-Flip (aka FADD-like apoptosis regulator; Cflar) as PU.1-regulated genes, and showed putative PU.1-binding sites in their proximal promoter regions.CASP8 and c-FLIP are known for their function downstream of death-receptor mediated apoptosis. Yet, it has been reported that CASP8 has non-apoptotic functions involving cell proliferation, differentiation, and inflammation. C-FLIP is the enzymatically inactive homolog of CASP8 and exists in three isoforms: a long isoform (c-FLIPL), that partially inhibits CASP8 activity, and two short forms (c-FLIPR and c-FLIPS) that are anti-apoptotic.To assess if CASP8 and/or c-FLIP are involved in PU.1-regulated cellular processes, we took advantage of the NB4 APL cell line model. These cells can be differentiated towards granulocytes with all-trans retinoic acid (ATRA) in a PU.1-dependent manner. We found an 8-10-fold induction of CASP8 and c-FLIP mRNA expression upon granulocytic differentiation of NB4 cells. Underlining a possible function of these two genes in granulocyte differentiation, we detected a markedly increased mRNA expression of both genes in human CD34+ hematopoietic cells differentiated towards neutrophils using G-CSF. Furthermore, knocking down PU.1 in NB4 cells significantly impaired CASP8 and cFLIP mRNA upregulation. Importantly, the anti-apoptotic cFLIPs isoform was exclusively induced after prolonged ATRA-treatment in PU.1 knockdown cells, whereas cFLIPL and CASP8 mRNA levels were reduced. The binding of PU.1 to the CFLAR promoter region together with altered cFLIP isoform ratio upon PU.1 expression indicates direct transcriptional activation of cFLIP and possibly an involvement of PU.1 in alternative splicing of cFLIP.Based on previous reports linking non-apoptotic CASP8 functions to macrophage differentiation and our findings of PU.1-dependent CASP8 regulation, we next studied the role of CASP8 in more detail during monocyte and macrophage differentiation by knocking down CASP8 expression in HL60 cells. HL60 CASP8 knockdown cells, generated using two independent shRNAs, were treated with vitamin D3 (VitD3) or PMA to induce monocyte or macrophage differentiation, respectively. Surprisingly, knocking down CASP8 led to increased CD11b expression together with increased pseudopodia formation. Furthermore, analysis of secreted cytokines in HL60 CASP8 knockdown cells suggests activation of macrophages towards an M2 phenotype.Our findings extend the role of PU.1 function to cell survival during granulocytic differentiation of APL cells. This occurs via distinct regulation of the pro-apoptotic CASP8, and anti-apoptotic cFLIP gene programs, respectively. Our findings suggest that increased expression of the anti-apoptotic, shorter cFLIP isoforms later in neutrophil differentiation may support short-term neutrophil cell survival. Lastly, our results implicate a novel PU.1-CASP8 pathway that may be necessary for alternative activation of M2 macrophages. DisclosuresNo relevant conflicts of interest to declare.

Synergistic Antitumour Properties of viscumTT in Alveolar Rhabdomyosarcoma.

Aqueous mistletoe extracts from the European mistletoe (Viscum album) contain mainly mistletoe lectins and viscotoxins as cytotoxic compounds. Lipophilic triterpene acids, which do not occur in conventional mistletoe preparations, were solubilised with β-cyclodextrins. The combination of an aqueous extract (viscum) and a triterpene-containing extract (TT) recreated a whole mistletoe extract (viscumTT). These extracts were tested on rhabdomyosarcoma in vitro, ex vivo, and in vivo with regard to anticancer effects. Viscum and viscumTT inhibited cell proliferation and induced apoptosis effectively in a dose-dependent manner in vitro and ex vivo, whereas TT showed only moderate inhibitory effects. viscumTT proved to be more effective than the single extracts and displayed a synergistic effect in vitro and a stronger effect in vivo. viscumTT induced apoptosis via the extrinsic and intrinsic pathways, evidenced by the loss of mitochondrial membrane potential and activation of CASP8 and CASP9. CASP10 inhibitor inhibited apoptosis effectively, emphasising the importance of CASP10 in viscumTT-induced apoptosis. Additionally, viscumTT changed the ratio of apoptosis-associated proteins by downregulation of antiapoptotic proteins such as XIAP and BIRC5, thus shifting the balance towards apoptosis. viscumTT effectively reduced tumour volume in patient-derived xenografts in vivo and may be considered a promising substance for rhabdomyosarcoma therapy.

Multiple Active Compounds from Viscum album L. Synergistically Converge to Promote Apoptosis in Ewing Sarcoma

Ewing sarcoma is the second most common bone cancer in children and adolescents, with poor prognosis and outcome in ~70% of initial diagnoses and 10–15% of relapses. Hydrophobic triterpene acids and hydrophilic lectins and viscotoxins from European mistletoe (Viscum album L.) demonstrate anticancer properties, but have not yet been investigated for Ewing sarcoma. Commercial Viscum album L. extracts are aqueous, excluding the insoluble triterpenes. We recreated a total mistletoe effect by combining an aqueous extract (viscum) and a triterpene extract (TT) solubilized with cyclodextrins. Ewing sarcoma cells were treated with viscum, TT and viscumTT in vitro, ex vivo and in vivo. In vitro and ex vivo treatment of Ewing sarcoma cells with viscum inhibited proliferation and induced apoptosis in a dose-dependent fashion, while viscumTT combination treatment generated a synergistic effect. Apoptosis occurred via intrinsic and extrinsic apoptotic pathways, evidenced by activation of both CASP8 and CASP9. We show that viscumTT treatment shifts the balance of apoptotic regulatory proteins towards apoptosis, mainly via CLSPN, MCL1, BIRC5 and XIAP downregulation. ViscumTT also demonstrated strong antitumor activity in a cell line- and patient-derived mouse model, and may be considered an adjuvant therapy option for pediatric patients with Ewing sarcoma.

Abstract 214: Balancing Innate Immunity Activation and Death Signals for Vascular Regeneration

Background: Our recent discoveries have shown that innate signaling supports effective nuclear reprogramming (Lee &amp; Sayed et al. Cell) and transdifferentiation to ECs (Sayed et al. Circ). By activating innate immunity (via TLR3), viral vectors used for reprogramming cause global epigenetic changes that favor an environment conducive for reprogramming. Indeed, activation of innate transcription factors (TFs) enhances EC regeneration, however, activation of the same pathway could induce: 1) apoptosis via activation of Casp8; and 2) programmed necrosis by receptor-interacting protein kinase 3 (RIP3), when Casp8 is absent. Hypothesis: We speculate that death pathways that are activated by these TFs compromise reprogramming. We previously showed that disruption of Casp8 led to mid-gestational death of mice due to unleashing of RIP3-dependent death pathways, preventing the formation of vascular endothelium and hematopoietic cells. By contrast, when Casp8 -/- mice were crossed with Rip3 -/- , the DKO mice developed normally with no stem cell defect. We hypothesize that these pathways exist as biological constraints on epigenetic plasticity and that pharmacological or genetic ablation could enhance EC reprogramming. Results: To test our hypothesis, we transdifferentiated WT and Casp8 -/- Rip3 -/- DKO MEFs using our protocol, employing an innate immunity modifier and EC growth factors. Intriguingly, our preliminary data showed that DKO MEFs transdifferentiated to induced-EC (iECs) with &gt;10-fold higher yield when compared to WT (yields ~30%). Genetic and functional assays showed that iECs generated from DKO MEFs were comparable to WT, indicating that removal of apoptotic pathways did not lead to aberrant reprogramming. Moreover, pharmacological inhibitors of death receptors when combined with our small molecule cocktail showed a similar increase in iEC generation in human fibroblasts. Conclusion: This study is a first step toward development of a regenerative strategy for PAD on the use of ECs derived from small molecules and growth factors without use of viral vectors encoding TFs. We intend to derive an effective and feasible technology for therapeutic transdifferentiation for ischemic syndromes to promote healing with tissue rather than a scar.

Remarkably intact antiviral CD8 T cell response despite the absence of caspase 8 apoptotic and RIP3 necrotic pathways (P1318)

Abstract Two programmed cell death pathways, apoptosis and necrosis, impact T cell function in mice and humans. Germ line disruption of pro-apoptotic caspase 8 (Casp8) activity in T cells triggers receptor interacting protein kinase (RIP)3-dependent necrosis upon TCR stimulation. This absolute requirement for Casp8 repression of RIP3-dependent pathways overshadows potential contributions of extrinsic apoptosis to T cell expansion and contraction. Mice deficient in both Casp8 and RIP3 (Casp8-/-Rip3-/-) have an intact immune compartment and maintain naïve CD8 T cells comparable to Rip3-/- and WT controls. Based on CD44 expression, aging mice show evidence of CD8 T cell proliferation and lymphadenopathy. Despite this, CD8 T cells in Casp8-/-Rip3-/- mice proliferate normally in response to TCR stimulation. Upon infection with murine CMV, Ag-specific CD8 T cells from Casp8-/-Rip3-/- mice expand, produce cytokines, kill cells expressing viral peptide, and control infection similarly to controls. Memory CD8 T cells develop in Casp8-/-Rip3-/- mice with appropriate KLRG1 and CD127 expression. These memory cells expand in response to and protect from secondary challenge. Additionally, memory inflation, a characteristic of the response to murine CMV, is preserved. Thus, when RIP3 pathways are eliminated, Casp8 restricts homeostatic lymphoproliferation but plays no direct role in the CD8 T cell response to infection.

FADD cleavage by NK cell granzyme M enhances its self-association to facilitate procaspase-8 recruitment for auto-processing leading to caspase cascade

Granzyme M (GzmM), an orphan Gzm, is constitutively and abundantly expressed in innate effector natural killer cells. We previously demonstrated that GzmM induces caspase (casp)-dependent apoptosis and cytochrome c release from mitochondria. We also resolved the crystal structure for GzmM and generated its specific inhibitor. However, how GzmM causes casp activation has not been defined. Here we found that casp-8 is an initiator caspase in GzmM-induced casp cascade, which causes other casp activation and Bid cleavage. GzmM does not directly cleave procaspase-3 and Bid, whose processing is casp dependent. Casp-8 knockdown or deficient cells attenuate or abolish GzmM-induced proteolysis of procaspase-3 and Bid. Extrinsic death receptor pathway adaptor Fas-associated protein with death domain (FADD) contributes to GzmM-induced casp-8 activation. GzmM specifically cleaves FADD after Met 196 to generate truncated FADD (tFADD) that enhances its self-association for oligomerization. The oligomerized tFADD facilitates procaspase-8 recruitment to promote its auto-processing leading to casp activation cascade. FADD-deficient cells abrogate GzmM-induced activation of casp-8 and apoptosis as well as significantly inhibit lymphokine-activated killer cell-mediated cytotoxicity. FADD processing by GzmM can potentiate killing efficacy against tumor cells and intracellular pathogens.

Complementary roles of Fas-associated death domain (FADD) and receptor interacting protein kinase-3 (RIPK3) in T-cell homeostasis and antiviral immunity

Caspase-8 (casp8) is required for extrinsic apoptosis, and mice deficient in casp8 fail to develop and die in utero while ultimately failing to maintain the proliferation of T cells, B cells, and a host of other cell types. Paradoxically, these failures are not caused by a defect in apoptosis, but by a presumed proliferative function of this protease. Indeed, following mitogenic stimulation, T cells lacking casp8 or its adaptor protein FADD (Fas-associated death domain protein) develop a hyperautophagic morphology, and die a programmed necrosis-like death process termed necroptosis. Recent studies have demonstrated that receptor-interacting protein kinases (RIPKs) RIPK1 and RIPK3 together facilitate TNF-induced necroptosis, but the precise role of RIPKs in the demise of T cells lacking FADD or casp8 activity is unknown. Here we demonstrate that RIPK3 and FADD have opposing and complementary roles in promoting T-cell clonal expansion and homeostasis. We show that the defective proliferation of T cells bearing an interfering form of FADD (FADDdd) is rescued by crossing with RIPK3(-/-) mice, although such rescue ultimately leads to lymphadenopathy. Enhanced recovery of these double-mutant T cells following stimulation demonstrates that FADD, casp8, and RIPK3 are all essential for clonal expansion, contraction, and antiviral responses. Finally, we demonstrate that caspase-mediated cleavage of RIPK1-containing necrosis inducing complexes (necrosomes) is sufficient to prevent necroptosis in the face of death receptor signaling. These studies highlight the "two-faced" nature of casp8 activity, promoting clonal expansion in some situations and apoptotic demise in others.

Enzymatically active single chain caspase-8 maintains T-cell survival during clonal expansion

The extrinsic, or death receptor, pathway integrates apoptotic signals through the protease caspase-8 (casp8). Beyond cell death regulation, non-apoptotic functions of casp8 include its essential requirement for hematopoiesis and lymphocyte clonal expansion, and tempering of autophagy in T cells. However, the mechanistic basis for the control of these disparate cellular processes remains elusive. Here, we show that casp8-deficient T-cell survival was rescued by enzymatically active, but not inactive, casp8-expressing retroviruses. The casp8 catalytic induction in proliferating T cell occurred independent of extrinsic and intrinsic apoptotic-signaling cascades and did not induce casp8 proteolytic processing. Using a biotinylated probe selectively targeting enzymatically active caspases, catalytically active full-length casp8 was found in vivo in dividing T cells. A casp8 D387A processing mutant was able to rescue casp8-deficient T-cell proliferation, validating that casp8 self-processing is not required for its non-apoptotic function(s). Finally, casp8 activity was highest in CD8(+) T cells, the most rapidly proliferating subset. These results show that the catalytically competent form of casp8 is required for rapid T-cell proliferation in response to TCR ligation, but that processing of the caspase is only necessary to promote apoptosis.

IFNγ Restores Breast Cancer Sensitivity to Fulvestrant by Regulating STAT1, IFN Regulatory Factor 1, NF-κB, BCL2 Family Members, and Signaling to Caspase-Dependent Apoptosis

Antiestrogens are effective therapies for the management of many estrogen receptor-alpha (ER)-positive breast cancers. Nonetheless, both de novo and acquired resistance occur and remain major problems in the clinical setting. IFNgamma is an inflammatory cytokine that induces the expression and function of IFN regulatory factor 1 (IRF1), a tumor suppressor gene that can increase antiestrogen responsiveness. We show that IFNgamma, but not IFNalpha, IFNbeta, or fulvestrant (ICI; ICI 182,780; Faslodex), induces IRF1 expression in antiestrogen-resistant MCF7/LCC9 and LY2 cells. Moreover, IFNgamma restores the responsiveness of these cells to fulvestrant. Increased IRF1 activation suppresses NF-kappaB p65 (RELA) activity, inhibits the expression of prosurvival (BCL2, BCL-W), and induces the expression of proapoptotic members (BAK, mitochondrial BAX) of the BCL2 family. This molecular signaling is associated with the activation of signal transducer and activator of transcription 1 and leads to increased mitochondrial membrane permeability; activation of caspase-7 (CASP7), CASP8, and CASP9; and induction of apoptosis but not autophagy. Whereas antiestrogen-resistant cells are capable of inducing autophagy through IFN-mediated signaling, their ability to do so through antiestrogen-regulated signaling is lost. The abilities of IFNgamma to activate CASP8, induce apoptosis, and restore antiestrogen sensitivity are prevented by siRNA targeting IRF1, whereas transient overexpression of IRF1 mimics the effects of IFNgamma treatment. These observations support the exploration of clinical trials combining antiestrogens and compounds that can induce IRF1, such as IFNgamma, for the treatment of some ER-positive breast cancers.

Non-apoptotic caspase-8 activation balances T lymphocyte autophagy (82.14)

Abstract Apoptosis and autophagy are critical mediators of a functional immune response. FADD and caspase-8 (casp8) were originally discovered to transduce apoptotic signals delivered through TNF family members. Paradoxically, lymphocytes lacking FADD or casp8 function have impaired clonal expansion following antigen receptor crosslinking and succumb to a caspase-independent cell death. We show that T cells lacking FADD or casp8 are subject to hyperactive autophagy. T cell autophagy induces casp8 activation through its interaction with FADD:Atg5:Atg12 complexes. Blockade of autophagic signaling with shRNA knockdown of the autophagy-dependent protein Atg7 leads to a rescue of T cells lacking FADD and casp8. Similarly, inhibition of RIPK1 kinase activity with necrostatin-1 completely restored T cell cycle and survival. We hypothesize that the non-apoptotic casp8 activity serves to dampen the autophagic response of proliferating T cells through direct cleavage of RIPK1 or the casp8 homolog, c-FLIP, which may secondarily inhibit RIPK1-dependent authophagic signaling. While rapidly dividing T cells require autophagy, this negative feedback loop can prevent hyperactive autophagy. Linking apoptotic proteins to this signaling paradigm may serve to prevent an inflammatory response driven by autophagic cell death in lymphocytes during an immune response. (Funded by NIH T32CA09054 to B.D.B. and R01AI63419 to C.M.W.)

Concurrent upregulation of endogenous proapoptotic and antiapoptotic factors in failing human hearts

Despite widespread activation of proapoptotic stimuli and mediators, the degree of apoptosis in failing hearts is not very high. Endogenous antiapoptotic mechanisms are thought to be triggered by the heart-failure process. We investigated whether activation of endogenous apoptosis inhibitors plays a part when death receptor and mitochondrial apoptotic pathways have been triggered. We evaluated various proapoptotic and antiapoptotic factors in myocardial tissue specimens obtained from normal and explanted end-stage ischemic and dilated cardiomyopathic hearts. Caspases (CASPs) 3, 8 and 9, total and activated Bcl-2 homology domain 3-interacting domain death agonist, the X-linked inhibitor of apoptosis (XIAP), and DNA fragmentation factor (DFF) proteins were analyzed by western blotting. Expression of messenger RNA was measured by reverse-transcription polymerase chain reaction for the XIAP, DIABLO, CFLAR and DFF genes. We also assessed CASP3, CASP8 and CASP9 and DFF activity. Cytochrome c1 localization in myocytes was analyzed by immunohistochemistry and immunoelectron microscopy. We collected myocardial tissue from eight cardiomyopathic hearts and five normal hearts. Cytochrome c1 was released from mitochondria into the cytosol in the cardiomyopathic hearts but CASP9 was not activated. CASP8 activity was increased compared with that in normal myocardium. Although CASP3 was cleaved, activity was not greatly increased because of an increase in XIAP and decrease in DIABLO expression. DFF proteins were conspicuously absent. Concurrent upregulation of endogenous antiapoptotic mechanisms can interrupt the apoptotic cascade and prevents cell loss despite the presence of multiple proapoptotic factors. This period might offer a therapeutic window for restoration of myocardial function in heart failure.

FADD and caspase-8 control the outcome of autophagic signaling in proliferating T cells

Fas-associated death domain protein (FADD) and caspase-8 (casp8) are vital intermediaries in apoptotic signaling induced by tumor necrosis factor family ligands. Paradoxically, lymphocytes lacking FADD or casp8 fail to undergo normal clonal expansion following antigen receptor cross-linking and succumb to caspase-independent cell death upon activation. Here we show that T cells lacking FADD or casp8 activity are subject to hyperactive autophagic signaling and subvert a cellular survival mechanism into a potent death process. T cell autophagy, enhanced by mitogenic signaling, recruits casp8 through interaction with FADD:Atg5-Atg12 complexes. Inhibition of autophagic signaling with 3-methyladenine, dominant-negative Vps34, or Atg7 shRNA rescued T cells expressing a dominant-negative FADD protein. The necroptosis inhibitor Nec-1, which blocks receptor interacting protein kinase 1 (RIP kinase 1), also completely rescued T cells lacking FADD or casp8 activity. Thus, while autophagy is necessary for rapid T cell proliferation, our findings suggest that FADD and casp8 form a feedback loop to limit autophagy and prevent this salvage pathway from inducing RIPK1-dependent necroptotic cell death. Thus, linkage of FADD and casp8 to autophagic signaling intermediates is essential for rapid T cell clonal expansion and may normally serve to promote caspase-dependent apoptosis under hyperautophagic conditions, thereby averting necrosis and inflammation in vivo.