Linda Roback

RIP3 Induces Apoptosis Independent of Pronecrotic Kinase Activity

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition

Significance The protein kinase receptor interacting protein 1 controls signaling via death receptors, Toll-like receptors, and retinoic acid-inducible gene 1-like receptors, dictating inflammatory outcomes as broad as cytokine activation and cell death. RIP1 makes a vital contribution during development, evident from the fact that RIP1-deficient mice die soon after birth. Here, we show that a kinase-independent function of RIP1 dampens the consequences of innate immune cell death. During parturition, RIP1 prevents the lethal consequences of RIP3-dependent necroptosis as well as caspase 8 (Casp8)-dependent apoptosis. In contrast to the RIP1-deficient phenotype, mice lacking a combination of RIP1, RIP3, and Casp8 are born and mature into viable, fertile, and immunocompetent adults. These results demonstrate the important protective role of RIP1 against physiologic and microbial death cues encountered at birth. The pronecrotic kinase, receptor interacting protein (RIP1, also called RIPK1) mediates programmed necrosis and, together with its partner, RIP3 (RIPK3), drives midgestational death of caspase 8 (Casp8)-deficient embryos. RIP1 controls a second vital step in mammalian development immediately after birth, the mechanism of which remains unresolved. Rip1−/− mice display perinatal lethality, accompanied by gross immune system abnormalities. Here we show that RIP1 K45A (kinase dead) knockin mice develop normally into adulthood, indicating that development does not require RIP1 kinase activity. In the face of complete RIP1 deficiency, cells develop sensitivity to RIP3-mixed lineage kinase domain-like–mediated necroptosis as well as to Casp8-mediated apoptosis activated by diverse innate immune stimuli (e.g., TNF, IFN, double-stranded RNA). When either RIP3 or Casp8 is disrupted in combination with RIP1, the resulting double knockout mice exhibit slightly prolonged survival over RIP1-deficient animals. Surprisingly, triple knockout mice with combined RIP1, RIP3, and Casp8 deficiency develop into viable and fertile adults, with the capacity to produce normal levels of myeloid and lymphoid lineage cells. Despite the combined deficiency, these mice sustain a functional immune system that responds robustly to viral challenge. A single allele of Rip3 is tolerated in Rip1−/−Casp8−/−Rip3+/− mice, contrasting the need to eliminate both alleles of either Rip1 or Rip3 to rescue midgestational death of Casp8-deficient mice. These observations reveal a vital kinase-independent role for RIP1 in preventing pronecrotic as well as proapoptotic signaling events associated with life-threatening innate immune activation at the time of mammalian parturition.

Caspase-8 scaffolding function and MLKL regulate NLRP3 inflammasome activation downstream of TLR3.

TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA.

Suppression of RIP3-dependent necroptosis by human cytomegalovirus.

Background: Viral suppressors of RHIM-dependent activation of pro-necrotic RIP3 kinase are crucial for successful infection in mice. Results: Human CMV blocks TNF-induced and murine CMV-induced necroptosis after RIP3 activation. Conclusion: Necrotic membrane leakage is blocked in infected cells despite the activation of MLKL. Significance: Viral inhibition of necroptosis will facilitate understanding of the final steps in this pathway. Necroptosis is an alternate programmed cell death pathway that is unleashed by caspase-8 compromise and mediated by receptor-interacting protein kinase 3 (RIP3). Murine cytomegalovirus (CMV) and herpes simplex virus (HSV) encode caspase-8 inhibitors that prevent apoptosis together with competitors of RIP homotypic interaction motif (RHIM)-dependent signal transduction to interrupt the necroptosis. Here, we show that pro-necrotic murine CMV M45 mutant virus drives virus-induced necroptosis during nonproductive infection of RIP3-expressing human fibroblasts, whereas WT virus does not. Thus, M45-encoded RHIM competitor, viral inhibitor of RIP activation, sustains viability of human cells like it is known to function in infected mouse cells. Importantly, human CMV is shown to block necroptosis induced by either TNF or M45 mutant murine CMV in RIP3-expressing human cells. Human CMV blocks TNF-induced necroptosis after RIP3 activation and phosphorylation of the mixed lineage kinase domain-like (MLKL) pseudokinase. An early, IE1-regulated viral gene product acts on a necroptosis step that follows MLKL phosphorylation prior to membrane leakage. This suppression strategy is distinct from RHIM signaling competition by murine CMV or HSV and interrupts an execution process that has not yet been fully elaborated.

The Human Cytomegalovirus UL36 Gene Controls Caspase-Dependent and -Independent Cell Death Programs Activated by Infection of Monocytes Differentiating to Macrophages

ABSTRACT The cellular protease caspase-8 activates extrinsic apoptosis and also functions to promote monocyte-to-macrophage differentiation. Differentiation-induced alterations to antiviral caspase-8-dependent cell death pathways are unclear. Here, we show THP-1 monocyte-to-macrophage differentiation alters the specific cell death pathways activated in response to human cytomegalovirus (HCMV) infection. Employing viruses with mutations in UL36, the gene that encodes the viral inhibitor of caspase-8 activation (vICA), our data indicate that both caspase-dependent and -independent death pathways are activated in response to infection. Activation of caspase-dependent and -independent cell death responses restricted growth of vICA-deficient viruses, and vICA/pUL36 inhibited either response. Thus, these studies also reveal that the UL36 gene controls a caspase-independent cell death pathway. The impact of caspases on control of antiviral responses differed at early and late stages of macrophage differentiation. Early in differentiation, vICA-deficient virus-induced cell death was dependent on caspases and inhibited by the pan-caspase inhibitor z-VAD(OMe)-fluoromethyl ketone. In contrast, virus-induced death at late times of differentiation was caspase independent. Additional unlabeled and fluorescent inhibitors indicated that caspase-8 promoted death from within infected cells at early but not late stages of differentiation. These data highlight the multifunctional role of vICA/pUL36 as HCMV encounters various antiviral responses during macrophage differentiation.

HtrA2/Omi terminates cytomegalovirus infection and is controlled by the viral mitochondrial inhibitor of apoptosis (vMIA).

Viruses encode suppressors of cell death to block intrinsic and extrinsic host-initiated death pathways that reduce viral yield as well as control the termination of infection. Cytomegalovirus (CMV) infection terminates by a caspase-independent cell fragmentation process after an extended period of continuous virus production. The viral mitochondria-localized inhibitor of apoptosis (vMIA; a product of the UL37x1 gene) controls this fragmentation process. UL37x1 mutant virus-infected cells fragment three to four days earlier than cells infected with wt virus. Here, we demonstrate that infected cell death is dependent on serine proteases. We identify mitochondrial serine protease HtrA2/Omi as the initiator of this caspase-independent death pathway. Infected fibroblasts develop susceptibility to death as levels of mitochondria-resident HtrA2/Omi protease increase. Cell death is suppressed by the serine protease inhibitor TLCK as well as by the HtrA2-specific inhibitor UCF-101. Experimental overexpression of HtrA2/Omi, but not a catalytic site mutant of the enzyme, sensitizes infected cells to death that can be blocked by vMIA or protease inhibitors. Uninfected cells are completely resistant to HtrA2/Omi induced death. Thus, in addition to suppression of apoptosis and autophagy, vMIA naturally controls a novel serine protease-dependent CMV-infected cell-specific programmed cell death (cmvPCD) pathway that terminates the CMV replication cycle.

Coupling of cAMP/PKA and MAPK signaling in neuronal cells is dependent on developmental stage

Neurite formation, an essential feature of neuronal development, is believed to involve participation of the ras-mitogen-activated protein kinase (MAPK) and cAMP-dependent protein kinase A (cAMP/PKA)-mediated signaling pathways. These pathways have been studied extensively in the rat pheochromocytoma cell line PC12, and current hypotheses suggest a single effector mechanism resulting from the convergence of cAMP/PKA and MAPK signaling. However, based on observations using a central neuronal progenitor cell line, AS583-8, there also exists evidence that the two signaling pathways may act independently resulting in neurites with differing dynamic features. In the present study, the upstream components of cAMP/PKA signaling were examined in AS583-8 cells as well as possible interactions with the MAPK pathway. We found that activation of PKA is both necessary and sufficient for the elaboration of rapidly forming processes, typical of the cAMP response. In addition, blockade of the MAPK pathway has no effect on the cAMP response, suggesting that activation of the cAMP/PKA pathway can stimulate neurite formation independent of the MAPK pathway. In order to evaluate which cell line model, PC12 vs AS583-8, best reflects the signaling features of developing central neurons, we examined interactions between cAMP/PKA and MAPK signaling in primary neuronal cultures from several brain regions. In immature cultures (1-day-old), at a point where the initiation of neurite formation is maximal, no interaction was observed. In more mature cultures (7 days old), where synaptic contacts have been established, we found a weak but reproducible activation of MAPK following stimulation of the cAMP/PKA pathway. These results suggest that cAMP/PKA and MAPK signaling act independently at the initiation of neuritogenesis but become coupled during later stages of neuronal development. Therefore, the interaction of the two pathways may be cell stage (younger vs older) specific and may participate in cellular functions that take place after initial neurite formation.

BETA -ADRENERGIC AND FIBROBLAST GROWTH FACTOR RECEPTORS INDUCE NEURONAL PROCESS OUTGROWTH THROUGH DIFFERENT MECHANISMS

The mechanisms that initiate and direct neuronal process formation remain poorly understood. We have recently described a neuronal progenitor cell line, AS583‐8.E4.22 (AS583‐8) which undergoes neurite formation in response to β2‐adrenergic and basic fibroblast growth factor (bFGF) receptor activation [Kwon, J.H et al. (1996) Eur. J. Neurosci., 8, 2042–2055]. In the present study, a comparison of these responses revealed that isoproterenol (ISO), a β‐adrenergic receptor agonist, induces multiple, highly branched processes within 30 min while bFGF induces fewer, unbranched processes within 24 h. In contrast to the ISO response, bFGF induces mitogen‐activated protein kinase activation and c‐fos expression in the cell line and results in neurite outgrowth that is dependent on new mRNA and protein synthesis. Two‐dimensional isoelectric focusing‐sodium dodecyl sulphate–polyacrylamide gel electrophoresis of cytoskeletal preparations revealed different patterns following ISO vs. bFGF exposure suggesting selective changes in protein expression and/or post‐translational modifications. Immunoblot analysis of these preparations for β‐tubulin, tyrosinated α‐tubulin and acetylated α‐tubulin also revealed different patterns following each type of treatment. Follow‐up confocal microscopy revealed that following ISO, the distribution of tyrosinated tubulin extends to the distal ends of processes whereas acetylated α‐tubulin is diminished within distal ends. This pattern has been reported to be associated with enhanced microtubule dynamics, a state in which process outgrowth is facilitated. In contrast, following bFGF treatment the distributions of tyrosinated and acetylated α‐tubulin were identical, a state associated with a diminution of microtubule dynamics. These results, a different time course of neurite formation, dependency on new gene expression and differential expression and cellular distribution of major cytoskeleton proteins suggest that neurite outgrowth induced by ISO vs. bFGF is mediated by two distinct intracellular effector mechanisms in AS583‐8 cells. In addition, studies, using the differential distribution of post‐translational modified α‐tubulins in neurites of primary neuronal cultures as marker for the two distinct processes of neurite formation suggest, that similar mechanisms are present in vivo. Therefore, the AS583‐8 cell line provides a useful model to study these signalling mechanisms that couple neurotransmitter and growth factor receptor activation to the cytoskeletal changes that mediate neurite formation.

Multiplicity-dependent activation of a serine protease-dependent cytomegalovirus-associated programmed cell death pathway

At a low MOI (≤0.01), cytomegalovirus-associated programmed cell death terminates productive infection via a pathway triggered by the mitochondrial serine protease HtrA2/Omi. This infected cell death is associated with late phase replication events naturally suppressed by the viral mitochondrial inhibitor of apoptosis (vMIA). Here, higher MOI (ranging from 0.1-3.0) triggers cell death earlier during infection independent of viral DNA synthesis. Thus, MOI-dependent activating signals early, at high MOI, or late, at low MOI, during replication promote serine protease-dependent death that is suppressed by vMIA. Treatment with an antioxidant targeting reactive oxygen species (ROS) or the serine protease inhibitor N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) delays cell death, and the combination has an additive impact. These studies identify serine proteases and ROS as important factors triggering programmed cell death induced by vMIA-deficient virus, and show that this death pathway occurs earlier and reduces viral yields as the MOI is increased.

Mouse cytomegalovirus M36 and M45 death suppressors cooperate to prevent inflammation resulting from antiviral programmed cell death pathways

Significance Caspase-8–mediated apoptotic and receptor-interacting protein (RIP)-dependent necroptotic signaling pathways are recognized host defense mechanisms that act by eliminating virus-infected cells. Cytomegalovirus-encoded inhibitors of apoptosis and necroptosis sustain infection and pathogenesis by preventing specific programmed cell death pathways. In the absence of viral inhibitors, combined apoptotic–necroptotic cell death signaling halts infection, preventing the virus from gaining a foothold in the host. We describe natural cooperation between apoptosis and necroptosis pathways in macrophages and within the host, resulting in robust proinflammatory cytokine responses not observed when infected cells die by either apoptosis or necroptosis alone. Thus, apoptosis combined with necroptosis serves a dual role in limiting herpesvirus persistence in the host. The complex interplay between caspase-8 and receptor-interacting protein (RIP) kinase RIP 3 (RIPK3) driving extrinsic apoptosis and necroptosis is not fully understood. Murine cytomegalovirus triggers both apoptosis and necroptosis in infected cells; however, encoded inhibitors of caspase-8 activity (M36) and RIP3 signaling (M45) suppress these antiviral responses. Here, we report that this virus activates caspase-8 in macrophages to trigger apoptosis that gives rise to secondary necroptosis. Infection with double-mutant ΔM36/M45mutRHIM virus reveals a signaling pattern in which caspase-8 activates caspase-3 to drive apoptosis with subsequent RIP3-dependent activation of mixed lineage kinase domain-like (MLKL) leading to necroptosis. This combined cell death signaling is highly inflammatory, greater than either apoptosis induced by ΔM36 or necroptosis induced by M45mutRHIM virus. IL-6 production by macrophages is dramatically increased during double-mutant virus infection and correlates with faster antiviral responses in the host. Collaboratively, M36 and M45 target caspase-8 and RIP3 pathways together to suppress this proinflammatory cell death. This study reveals the effect of antiviral programmed cell death pathways on inflammation, shows that caspase-8 activation may go hand-in-hand with necroptosis in macrophages, and revises current understanding of independent and collaborative functions of M36 and M45 in blocking apoptotic and necroptotic cell death responses.