Human SV80 Cells: Control vs. Caspase-8 knock down
ABSTRACT: Excessive responses to pattern-recognition receptors are prevented by regulatory mechanisms that affect the amounts, conformation, and associative properties of their signaling proteins. We report that signaling by the ribonucleic acid sensor RIG-I is restricted, in addition, by caspase-mediated cleavage that results in conversion of a signaling enhancer to a signaling inhibitor. RIP1 and caspase-8, two proteins known to mediate effects of receptors of the TNF/NGF family, are recruited to the RIG-I complex following viral infection, and serve in a coordinated manner antagonistic regulatory roles: conjugation of a ubiquitin chain to Lys-377 in RIP1 facilitates assembly of the RIG-I complex, but it also renders RIP1 susceptible to caspase-8-mediated cleavage, yielding an inhibitory RIP1 fragment. The dependence of RIP1 cleavage on the same molecular change as the one that facilitates RIG-I signaling allows for RIG-I signaling to be restricted in its duration without compromising its initial activation. Transcriptional profiling of human SV80 cells comparing cells infected with control lenti virus to cells infected with caspase-8 siRNA expressing lenti virus. Goal was to determine the effects of caspase-8 knock down on global gene expression. Two-condition experiment, Control lenti Vs caspase-8 siRNA expressing lenti. Biological replicates: 4 control, 4 caspase-8 siRNA infected
Project description:Excessive responses to pattern-recognition receptors are prevented by regulatory mechanisms that affect the amounts, conformation, and associative properties of their signaling proteins. We report that signaling by the ribonucleic acid sensor RIG-I is restricted, in addition, by caspase-mediated cleavage that results in conversion of a signaling enhancer to a signaling inhibitor. RIP1 and caspase-8, two proteins known to mediate effects of receptors of the TNF/NGF family, are recruited to the RIG-I complex following viral infection, and serve in a coordinated manner antagonistic regulatory roles: conjugation of a ubiquitin chain to Lys-377 in RIP1 facilitates assembly of the RIG-I complex, but it also renders RIP1 susceptible to caspase-8-mediated cleavage, yielding an inhibitory RIP1 fragment. The dependence of RIP1 cleavage on the same molecular change as the one that facilitates RIG-I signaling allows for RIG-I signaling to be restricted in its duration without compromising its initial activation. Overall design: Transcriptional profiling of human SV80 cells comparing cells infected with control lenti virus to cells infected with caspase-8 siRNA expressing lenti virus. Goal was to determine the effects of caspase-8 knock down on global gene expression. Two-condition experiment, Control lenti Vs caspase-8 siRNA expressing lenti. Biological replicates: 4 control, 4 caspase-8 siRNA infected
Project description:The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TNFSF12, CD255) (TWEAK) can stimulate apoptosis in certain cancer cells. Previous studies suggest that TWEAK activates cell death indirectly, by inducing TNF?-mediated autocrine signals. However, the underlying death-signaling mechanism has not been directly defined. Consistent with earlier work, TWEAK assembled a proximal signaling complex containing its cognate receptor FN14, the adaptor TRAF2, and cellular inhibitor of apoptosis protein 1 (cIAP1). Neither the death domain adaptor Fas-associated death domain nor the apoptosis-initiating protease caspase-8 associated with this primary complex. Rather, TWEAK induced TNF? secretion and TNF receptor 1-dependent assembly of a death-signaling complex containing receptor-interacting protein 1 (RIP1), FADD, and caspase-8. Knockdown of RIP1 by siRNA prevented TWEAK-induced association of FADD with caspase-8 but not formation of the FN14-TRAF2-cIAP1 complex and inhibited apoptosis activation. Depletion of the RIP1 E3 ubiquitin ligase cIAP1 enhanced assembly of the RIP1-FADD-caspase-8 complex and augmented cell death. Conversely, knockdown of the RIP1 deubiquitinase CYLD inhibited these functions. Depletion of FADD, caspase-8, BID, or BAX and BAK but not RIP3 attenuated TWEAK-induced cell death. Pharmacologic inhibition of the NF-?B pathway or siRNA knockdown of RelA attenuated TWEAK induction of TNF? and association of RIP1 with FADD and caspase-8. These results suggest that TWEAK triggers apoptosis by promoting assembly of a RIP1-FADD-caspse-8 complex via autocrine TNF?-TNFR1 signaling. The proapoptotic activity of TWEAK is modulated by cIAP1 and CYLD and engages both the extrinsic and intrinsic signaling pathways.
Project description:Newcastle disease virus (NDV) is an oncolytic virus which selectively replicates in tumor cells and exerts anti-tumor cytotoxic activity by promoting cell death. In this study, we focus on characterization of the underlying mechanisms of NDV-induced cell death in HeLa cells. We find that NDV Herts/33 strain triggers both extrinsic and intrinsic apoptosis at late infection times. The activation of NF-ÐºB pathway and subsequent up-regulation of TNF-?/TRAIL initiates extrinsic apoptosis, leading to activation of caspase 8 and cleavage of Bid into tBid. tBid transmits the extrinsic apoptotic signals to mitochondria and mediates intrinsic apoptosis, which is hallmarked by cleavage of caspase 9. Moreover, RIP1 is cleaved into RIP1-N and RIP1-C at D324 by caspase 8, and this cleavage promotes apoptosis. Surprisingly, over expression of RIP1 reduces apoptosis and depletion of RIP1 promotes apoptosis, suggesting full length RIP1 is anti-apoptotic. Moreover, necroptosis hallmark protein MLKL is activated by phosphorylation at 12-24 h.p.i., and RIP1 regulates the level of phosphor-MLKL. Immunostaining shows that RIP1 aggregates to stress granules (SGs) at 8-24 h.p.i., and phosphor-MLKL is also recruited to SGs, instead of migrating to plasma membrane to exert its necrotic function. Immunoprecipitation study demonstrates that RIP1 bind to phosphor-MLKL, and depletion of RIP1 reduces the aggregation of MLKL to SGs, suggesting that RIP1 recruits MLKL to SGs. Altogether, NDV infection initiates extrinsic apoptosis via activation of NF-ÐºB and secretion of TNF-?/TRAIL. Activation of caspase 8 by TNF-?/TRAIL and subsequent cleavage of Bid and RIP1 transmit the death signals to mitochondria. Meanwhile, virus subverts the host defensive necroptosis via recruiting phosphor-MLKL by RIP1 to SGs. Thus, RIP1 is a central signaling protein in regulation of apoptosis and necroptosis during NDV infection.
Project description:Although TRAIL is considered a potential anticancer agent, it enhances tumor progression by activating NF-?B in apoptosis-resistant cells. Cellular FLICE-like inhibitory protein (cFLIP) overexpression and caspase-8 activation have been implicated in TRAIL-induced NF-?B activation; however, the underlying mechanisms are unknown. Here, we report that caspase-8-dependent cleavage of RIP1 in the kinase domain (KD) and intermediate domain (ID) determines the activation state of the NF-?B pathway in response to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. In apoptosis-sensitive cells, caspase-8 cleaves RIP1 in the KD and ID immediately after the recruitment of RIP1 to the receptor complex, impairing I?B kinase (IKK) recruitment and NF-?B activation. In apoptosis-resistant cells, cFLIP restricts caspase-8 activity, resulting in limited RIP1 cleavage and generation of a KD-cleaved fragment capable of activating NF-?B but not apoptosis. Notably, depletion of the cytoplasmic pool of TRAF2 and cIAP1 in lymphomas by CD40 ligation inhibits basal RIP1 ubiquitination but does not prompt cell death, due to CD40L-induced cFLIP expression and limited RIP1 cleavage. Inhibition of RIP1 cleavage at the KD suppresses NF-?B activation and cell survival even in cFLIP-overexpressing lymphomas. Importantly, RIP1 is constitutively cleaved in human and mouse lymphomas, suggesting that cFLIP-mediated and caspase-8-dependent limited cleavage of RIP1 is a new layer of mechanism that promotes NF-?B activation and lymphoma survival.
Project description:Endoplasmic reticulum (ER) stress contributes to the development and progression of many chronic inflammatory diseases, including type 2 diabetes, obesity, atherosclerosis, neurodegenerative diseases, and cancer. ER stress has been reported to induce inflammasome activation and release of mature IL-1?, which contributes to many inflammatory diseases. The molecular mechanisms that activate the inflammasome during ER stress are still poorly understood. Here we report that the kinase receptor-interacting protein 1 (RIP1) plays an important role in ER stress-induced activation of inflammasome. Inhibition of RIP1 kinase activity by Necrostatin-1 or siRNA-mediated RIP1 knockdown significantly reduced ER stress-induced caspase-1 cleavage and IL-1? secretion in both bone marrow-derived macrophages (BMDMs) and J774A.1 macrophages. We speculate that the mitochondria fission factor dynamin-related protein 1 (DRP1) and reactive oxygen species (ROS) might function as the effectors downstream of RIP1 to mediate inflammasome activation. Our study reveals a critical role for RIP1 in regulating ER stress-induced inflammation responses, and proposes RIP1 as a potential pharmaceutical target to treat diseases resulting from unresolved ER stress-related inflammation.
Project description:Death receptor-induced programmed necrosis is regarded as a secondary death mechanism dominating only in cells that cannot properly induce caspase-dependent apoptosis. Here, we show that in cells lacking TGF?-activated Kinase-1 (TAK1) expression, catalytically active Receptor Interacting Protein 1 (RIP1)-dependent programmed necrosis overrides apoptotic processes following Tumor Necrosis Factor-? (TNF?) stimulation and results in rapid cell death. Importantly, the activation of the caspase cascade and caspase-8-mediated RIP1 cleavage in TNF?-stimulated TAK1 deficient cells is not sufficient to prevent RIP1-dependent necrosome formation and subsequent programmed necrosis. Our results demonstrate that TAK1 acts independently of its kinase activity to prevent the premature dissociation of ubiquitinated-RIP1 from TNF?-stimulated TNF-receptor I and also to inhibit the formation of TNF?-induced necrosome complex consisting of RIP1, RIP3, FADD, caspase-8 and cFLIP(L). The surprising prevalence of catalytically active RIP1-dependent programmed necrosis over apoptosis despite ongoing caspase activity implicates a complex regulatory mechanism governing the decision between both cell death pathways following death receptor stimulation.
Project description:Tanshinone IIA (Tan IIA), a constituent of the traditional medicinal plant Salvia miltiorrhiza BUNGE, has been reported to possess anticancer activity through induction of apoptosis in many cancer cells. Surprisingly, the present study finds that Tan IIA simultaneously causes apoptosis and necroptosis in human hepatocellular carcinoma HepG2 cells. We further find that apoptosis can be converted to necroptosis by pan-caspase inhibitor Z-VAD-fmk, and the two death modes can be blocked by necroptotic inhibitor necrostatin-1. The underlying mechanisms are revealed by analysis of the signaling molecules using western blotting. In control cells, FLICE inhibitory protein in short form (FLIPS) is expressed in relatively high levels and binds to caspase 8 in ripoptosome, which supposedly sustains cell survival. However, in Tan IIA-treated cells, FLIPS is down-regulated and may thus cause homodimer formation of cleaved caspase 8, cleavage of receptor-interacting serine/threonine-protein kinases 1, 3 (RIP1, RIP3), and mixed-lineage kinase domain-like (MLKL), in turn leads to cell apoptosis. In parallel, Tan IIA causes necroptosis by forming a suggested necrosomal complex composed of RIP1/RIP3. Regarding the inhibitors, z-VAD-fmk diminishes the cleaved caspase 8, RIP1, RIP3, and MLKL induced by Tan IIA, and reconstructs the ripoptosome complex, which marks cells moving from apoptosis to necroptosis. Nec-1 recovers the Tan IIA down-regulated FLIPS, consequently causes FLIPS to form heterodimer with caspase 8 and thus block apoptosis. Meanwhile, cleaved forms of RIP1 and RIP3 were observed preventing necroptosis. Intriguingly, the cytotoxicity of tumor necrosis factor-related apoptosis-inducing ligand to HepG2 cells is enhanced by Tan IIA in a pilot study, which may be attributed to low FLIPS levels induced by Tan IIA. In short, Tan IIA simultaneously induces both Nec-1 inhibition and FLIPS regulation-mediated apoptosis/necroptosis, which has not been previously documented. Moreover, the involvement of the cleavage type of MLKL in executing necroptosis warrants further investigation.
Project description:Junin virus (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF), a human disease with a high case-fatality rate. It is widely accepted that arenaviral infections, including JUNV infections, are generally non-cytopathic. In contrast, here we demonstrated apoptosis induction in human lung epithelial carcinoma (A549), human hepatocarcinoma and Vero cells upon infection with the attenuated Candid#1 strain of, JUNV as determined by phosphatidylserine (PS) translocation, Caspase 3 (CASP3) activation, Poly (ADP-ribose) polymerase (PARP) cleavage and/or chromosomal DNA fragmentation. Moreover, as determined by DNA fragmentation, we found that the pathogenic Romero strain of JUNV was less cytopathic than Candid#1 in human hepatocarcinoma and Vero, but more apoptotic in A549 and Vero E6 cells. Additionally, we found that JUNV-induced apoptosis was enhanced by RIG-I signaling. Consistent with the previously reported role of RIG-I like helicase (RLH) signaling in initiating programmed cell death, we showed that cell death or DNA fragmentation of Candid#1-infected A549 cells was decreased upon siRNA or shRNA silencing of components of RIG-I pathway in spite of increased virus production. Similarly, we observed decreased DNA fragmentation in JUNV-infected human hepatocarcinoma cells deficient for RIG-I when compared with that of RIG-I-competent cells. In addition, DNA fragmentation detected upon Candid#1 infection of type I interferon (IFN)-deficient Vero cells suggested a type I IFN-independent mechanism of apoptosis induction in response to JUNV. Our work demonstrated for the first time apoptosis induction in various cells of mammalian origin in response to JUNV infection and partial mechanism of this cell death.
Project description:RIP1 is a serine/threonine kinase, which is involved in apoptosis and necroptosis. In apoptosis, caspase-8 and FADD have an important role. On the other hand, RIP3 is a key molecule in necroptosis. Recently, we reported that eleostearic acid (ESA) elicits caspase-3- and PARP-1-independent cell death, although ESA-treated cells mediate typical apoptotic morphology such as chromatin condensation, plasma membrane blebbing and apoptotic body formation. The activation of caspases, Bax and PARP-1, the cleavage of AIF and the phosphorylation of histone H2AX, all of which are characteristics of typical apoptosis, do not occur in ESA-treated cells. However, the underlying mechanism remains unclear. To clarify the signaling pathways in ESA-mediated apoptosis, we investigated the functions of RIP1, MEK, ERK, as well as AIF. Using an extensive study based on molecular biology, we identified the alternative role of RIP1 in ESA-mediated apoptosis. ESA mediates RIP1-dependent apoptosis in a kinase independent manner. ESA activates serine/threonine phosphatases such as calcineurin, which induces RIP1 dephosphorylation, thereby ERK pathway is activated. Consequently, localization of AIF and ERK in the nucleus, ROS generation and ATP reduction in mitochondria are induced to disrupt mitochondrial cristae, which leads to cell death. Necrostatin (Nec)-1 blocked MEK/ERK phosphorylation and ESA-mediated apoptosis. Nec-1 inactive form (Nec1i) also impaired ESA-mediated apoptosis. Nec1 blocked the interaction of MEK with ERK upon ESA stimulation. Together, these findings provide a new finding that ERK and kinase-independent RIP1 proteins are implicated in atypical ESA-mediated apoptosis.
Project description:Necroptosis has emerged as a new form of programmed cell death implicated in a number of pathological conditions such as ischemic injury, neurodegenerative disease, and viral infection. Recent studies indicate that TGF?-activated kinase 1 (TAK1) is nodal regulator of necroptotic cell death, although the underlying molecular regulatory mechanisms are not well defined. Here we reported that TAK1 regulates necroptotic signaling as well as caspase 8-mediated apoptotic signaling through both NF?B-dependent and -independent mechanisms. Inhibition of TAK1 promoted TNF?-induced cell death through the induction of RIP1 phosphorylation/activation and necrosome formation. Further, inhibition of TAK1 triggered two caspase 8 activation pathways through the induction of RIP1-FADD-caspase 8 complex as well as FLIP cleavage/degradation. Mechanistically, our data uncovered an essential role for the adaptor protein TNF receptor-associated protein with death domain (TRADD) in caspase 8 activation and necrosome formation triggered by TAK1 inhibition. Moreover, ablation of the deubiqutinase CYLD prevented both apoptotic and necroptotic signaling induced by TAK1 inhibition. Finally, blocking the ubiquitin-proteasome pathway prevented the degradation of key pro-survival signaling proteins and necrosome formation. Thus, we identified new regulatory mechanisms underlying the critical role of TAK1 in cell survival through regulation of multiple cell death checkpoints. Targeting key components of the necroptotic pathway (e.g., TRADD and CYLD) and the ubiquitin-proteasome pathway may represent novel therapeutic strategies for pathological conditions driven by necroptosis.