Vladimir Tchikov

Regulation of TNFR1 and CD95 signalling by receptor compartmentalization

The death receptors tumour-necrosis factor receptor-1 (TNFR1) and CD95 (also known as FAS and APO-1) transduce signals that promote cell death by apoptosis. However, these receptors are also capable of inducing anti-apoptotic signals through the activation of the transcription factor nuclear factor-κB (NF-κB) or through activation of the proliferative mitogen-activated protein kinase (MAPK) cascade. Recent findings reveal a role for receptor internalization and endosomal trafficking in selectively transmitting the signals that lead either to apoptosis or to the survival of the cell.

The role of receptor internalization in CD95 signaling

Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death‐inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand‐mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF‐κB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates.

Riboflavin kinase couples TNF receptor 1 to NADPH oxidase

Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to p22phox, the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and p22phox is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase.

Palmitoylation of CD95 facilitates formation of SDS‐stable receptor aggregates that initiate apoptosis signaling

Apoptosis signaling through CD95 (Fas/APO‐1) involves aggregation and clustering of the receptor followed by its actin‐dependent internalization. Internalization is required for efficient formation of the death‐inducing signaling complex (DISC) with maximal recruitment of FADD, caspase‐8/10 and c‐FLIP occurring when the receptor has reached an endosomal compartment. The first detectable event during CD95 signaling is the formation of SDS‐stable aggregates likely reflecting intense oligomerization of the receptor. We now demonstrate that these SDS‐stable forms of CD95 correspond to very high molecular weight DISC complexes (hiDISC) and are the sites of caspase‐8 activation. hiDISCs are found both inside and outside of detergent‐resistant membranes. The formation of SDS‐stable CD95 aggregates involves palmitoylation of the membrane proximal cysteine 199 in CD95. Cysteine 199 mutants no longer form SDS‐stable aggregates, and inhibition of palmitoylation reduces internalization of CD95 and activation of caspase‐8. Our data demonstrate that SDS‐stable forms of CD95 are the sites of apoptosis initiation and represent an important early step in apoptosis signaling through CD95 before activation of caspases.

Inhibition of TNF receptor 1 internalization by adenovirus 14.7K as a novel immune escape mechanism

The adenoviral protein E3-14.7K (14.7K) is an inhibitor of TNF-induced apoptosis, but the molecular mechanism underlying this protective effect has not yet been explained exhaustively. TNF-mediated apoptosis is initiated by ligand-induced recruitment of TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), and caspase-8 to the death domain of TNF receptor 1 (TNFR1), thereby establishing the death-inducing signaling complex (DISC). Here we report that adenovirus 14.7K protein inhibits ligand-induced TNFR1 internalization. Analysis of purified magnetically labeled TNFR1 complexes from murine and human cells stably transduced with 14.7K revealed that prevention of TNFR1 internalization resulted in inhibition of DISC formation. In contrast, 14.7K did not affect TNF-induced NF-kappaB activation via recruitment of receptor-interacting protein 1 (RIP-1) and TNF receptor-associated factor 2 (TRAF-2). Inhibition of endocytosis by 14.7K was effected by failure of coordinated temporal and spatial assembly of essential components of the endocytic machinery such as Rab5 and dynamin 2 at the site of the activated TNFR1. Furthermore, we found that the same TNF defense mechanisms were instrumental in protecting wild-type adenovirus-infected human cells expressing 14.7K. This study describes a new molecular mechanism implemented by a virus to escape immunosurveillance by selectively targeting TNFR1 endocytosis to prevent TNF-induced DISC formation.

Caspase‐8 and caspase‐7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF‐R1 receptosomes

We previously demonstrated that tumour necrosis factor (TNF)‐induced ceramide production by endosomal acid sphingomyelinase (A‐SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase‐9 and caspase‐3 activation. The mechanism of TNF‐mediated A‐SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF‐induced A‐SMase activation depends on functional caspase‐8 and caspase‐7 expression. The active forms of all three enzymes, caspase‐8, caspase‐7 and A‐SMase, but not caspase‐3, colocalize in internalized TNF receptosomes. While caspase‐8 and caspase‐3 are unable to induce activation of purified pro‐A‐SMase, we found that caspase‐7 mediates A‐SMase activation by direct interaction resulting in proteolytic cleavage of the 72‐kDa pro‐A‐SMase zymogen at the non‐canonical cleavage site after aspartate 253, generating an active 57 kDa A‐SMase molecule. Caspase‐7 down modulation revealed the functional link between caspase‐7 and A‐SMase, confirming proteolytic cleavage as one further mode of A‐SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase‐8 and caspase‐7 for induction of A‐SMase activation by proteolytic cleavage of pro‐A‐SMase.

TRAIL signaling is mediated by DR4 in pancreatic tumor cells despite the expression of functional DR5

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and agonistic anti-DR4/TRAIL-R1 and anti-DR5/TRAIL-R2 antibodies are currently under clinical investigation for treatment of different malignancies. TRAIL activates DR4 and DR5 and thereby triggers apoptotic and non-apoptotic signaling pathways, but possible different roles of DR4 or DR5 in these responses has poorly been addressed so far. In the present work, we analyzed cell viability, DISC formation as well as IL-8 and NF–κB activation side by side in responses to TRAIL and agonistic antibodies against DR4 (mapatumumab) and against DR5 (lexatumumab) in pancreatic ductal adenocarcinoma cells. We found that all three reagents are able to activate cell death and pro-inflammatory signaling. Death-inducing signaling complex (DISC) analysis revealed that mapatumumab and lexatumumab induce formation of homocomplexes of either DR4 or DR5, whereas TRAIL additionally stimulated the formation of heterocomplexes of both receptors. Notably, blocking of receptors using DR4- and DR5-specific Fab fragments indicated that TRAIL exerted its function predominantly via DR4. Interestingly, inhibition of PKC by Goe6983 enabled DR5 to trigger apoptotic signaling in response to TRAIL and also strongly enhanced lexatumumab-mediated cell death. Our results suggest the existence of mechanisms that silence DR5 for TRAIL- but not for agonistic-antibody treatment.

CARP-2 Is an Endosome-Associated Ubiquitin Ligase for RIP and Regulates TNF-Induced NF-κB Activation

BACKGROUNDnThe proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) elicits cellular responses by signaling through a receptor complex that includes the essential adaptor molecule RIP. One important consequence of signaling is activation of the transcription factor NF-kappaB, and failure to downregulate TNF-induced NF-kappaB transcriptional activity results in chronic inflammation and death. Internalization of the receptor complex plays an important regulatory role in TNF signaling.nnnRESULTSnWe report that CARP-2, a RING domain-containing ubiquitin protein ligase (E3), is a negative regulator of TNF-induced NF-kappaB activation. By virtue of its phospholipid-binding FYVE domain, CARP-2 localized to endocytic vesicles, where it interacted with internalized TNF-receptor complex, resulting in RIP ubiquitination and degradation. Knockdown of CARP-2 stabilized TNFR1-associated polyubiquitinated RIP levels after TNF simulation and enhanced activation of NF-kappaB.nnnCONCLUSIONSnCARP-2 acts at the level of endocytic vesicles to limit the intensity of TNF-induced NF-kappaB activation by the regulated elimination of a necessary signaling component within the receptor complex.

The Polycomb group protein EED couples TNF receptor 1 to neutral sphingomyelinase

The phospholipase neutral sphingomyelinase (N-SMase) has been recognized as a major mediator of processes such as inflammation, development and growth, differentiation and death of cells, as well as in diseases such as Alzheimer’s, atherosclerosis, heart failure, ischemia/reperfusion damage, or combined pituitary hormone deficiency. Although activation of N-SMase by the proinflammatory cytokine TNF was described almost two decades ago, the underlying signaling pathway is unresolved. Here, we identify the Polycomb group protein EED (embryonic ectodermal development) as an interaction partner of nSMase2. In yeast, the N terminus of EED binds to the catalytic domain of nSMase2 as well as to RACK1, a protein that modulates the activation of nSMase2 by TNF in concert with the TNF receptor 1 (TNF-R1)-associated protein FAN. In mammalian cells, TNF causes endogenous EED to translocate from the nucleus and to colocalize and physically interact with both endogenous nSMase2 and RACK1. As a consequence, EED and nSMase2 are recruited to the TNF-R1•FAN•RACK1-complex in a timeframe concurrent with activation of nSMase2. After knockdown of EED by RNA interference, the TNF-dependent activation of nSMase2 is completely abrogated, identifying EED as a protein that both physically and functionally couples TNF-R1 to nSMase2, and which therefore represents the “missing link” that completes one of the last unresolved signaling pathways of TNF-R1.

Subcellular compartmentalization of TNF receptor-1 and CD95 signaling pathways.

Receptors that belong to the family of death-receptors including TNF receptor-1 (TNF-R1), CD95 (Fas, APO-1) and TRAIL receptors (TRAIL-R1, TRAIL R2/DR4/DR5) transduce signals resulting in entirely different biological outcomes: They promote cell death via apoptosis but are also capable of inducing anti-apoptotic signals through the transcription factor nuclear factor NF-κB or activation of the proliferative MAPK/ERK protein kinase cascade resulting in cell protection and tissue regeneration. Recent findings revealed a regulatory role of receptor internalization and its intracellular trafficking in selectively transmitting signals that lead either to apoptosis or to the survival of the cell, providing a clue to the understanding of these contradictory biological phenomena. In this chapter we review our data obtained during the Collaborative Research Center 415 (CRC 415) focusing on the compartmentalization of TNF-R1 and CD95 pro and anti-apoptotic signaling. We will address the role of internalization in determining the fate of the receptors. We suggest that fusion of internalized TNF-receptosomes with trans-Golgi vesicles is a novel mechanism to transduce death signals along the endosomal trafficking route. The roles of acid sphingomyelinase, the lipid second messenger ceramide, and the aspartate-protease cathepsin D as novel players in the cell death scenario is also highlighted. We report on the regulation of NF-κB signaling by recruitment of the endosomal E3-ubiquitin ligases CARP-2 and CARP-1 during TNF-receptosome trafficking. The biological significance of TNF receptor-1 compartmentalization is demonstrated by the strategy of adenoviruses to impede TNF-R1 internalization and by this preventing host cell apoptosis.