A role for c-FLIPL in the regulation of apoptosis,autophagy, and necroptosis in T lymphocytes

Caspase 8 plays a dual role in the survival of T lymphocytes. Although active caspase 8 mediates apoptosis upon death receptor signaling, the loss of caspase 8 activity leads to receptor-interacting protein (RIP)-1/RIP-3-dependent necrotic cell death (necroptosis) upon TCR activation. The anti-apoptotic protein c-FLIP (cellular caspase 8 (FLICE)-like inhibitory protein) suppresses death receptor-induced caspase 8 activation. Moreover, recent findings suggest that c-FLIP is also involved in inhibiting necroptosis and autophagy. It remains unclear whether c-FLIP protects primary T lymphocytes from necroptosis or regulates the threshold at which autophagy occurs. Here, we used a c-FLIP isoform-specific conditional deletion model to show that c-FLIP_L-deficient T cells underwent RIP-1-dependent necroptosis upon TCR stimulation. Interestingly, although previous studies have only described necroptosis in the absence of caspase 8 activity, we found that pro-apoptotic caspase 8 activity and apoptosis were also enhanced in c-FLIP_L-deficient T lymphocytes. Furthermore, c-FLIP_L-deficient T cells exhibited enhanced autophagy, which served a cytoprotective function. Together, these findings indicate that c-FLIP_L plays an important antinecroptotic role and is a key regulator of apoptosis, autophagy, and necroptosis in T lymphocytes.     

Participation of c-FLIP in NLRP3 and AIM2 inflammasome activation

Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage survival. Recent studies have revealed a selective role of caspase-8 in noncanonical IL-1β production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIP_L is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1β. Hemizygotic deletion of c-FLIP impaired ATP- and monosodium uric acid (MSU)-induced IL-1β production in macrophages primed through Toll-like receptors (TLRs). Decreased IL-1β expression was attributed to a reduced activation of caspase-1 in c-FLIP hemizygotic cells. In contrast, the production of TNF-α was not affected by downregulation in c-FLIP. c-FLIP_L interacted with NLRP3 or procaspase-1. c-FLIP is required for the full NLRP3 inflammasome assembly and NLRP3 mitochondrial localization, and c-FLIP is associated with NLRP3 inflammasome. c-FLIP downregulation also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic-, FasL-, or Dectin-1-induced IL-1β generation that is caspase-8-mediated. Our results demonstrate a prominent role of c-FLIP_L in the optimal activation of the NLRP3 and AIM2 inflammasomes, and suggest that c-FLIP could be a valid target for treatment of inflammatory diseases caused by over-activation of inflammasomes.     

Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL

Cellular FADD-like interleukin-1β–converting enzyme inhibitory proteins (c-FLIPs; isoforms c-FLIP long [c-FLIP_L], c-FLIP short [c-FLIP_S], and c-FLIP Raji [c-FLIP_R]) regulate caspase-8 activation and death receptor (DR)–induced apoptosis. In this study, using a combination of mathematical modeling, imaging, and quantitative Western blots, we present a new mathematical model describing caspase-8 activation in quantitative terms, which highlights the influence of c-FLIP proteins on this process directly at the CD95 death-inducing signaling complex. We quantitatively define how the stoichiometry of c-FLIP proteins determines sensitivity toward CD95-induced apoptosis. We show that c-FLIP_L has a proapoptotic role only upon moderate expression in combination with strong receptor stimulation or in the presence of high amounts of one of the short c-FLIP isoforms, c-FLIP_S or c-FLIP_R. Our findings resolve the present controversial discussion on the function of c-FLIP_L as a pro- or antiapoptotic protein in DR-mediated apoptosis and are important for understanding the regulation of CD95-induced apoptosis, where subtle differences in c-FLIP concentrations determine life or death of the cells.     

The role of c-FLIP splice variants in urothelial tumours

Deregulation of apoptosis is common in cancer and is often caused by overexpression of anti-apoptotic proteins in tumour cells. One important regulator of apoptosis is the cellular FLICE-inhibitory protein (c-FLIP), which is overexpressed, for example, in melanoma and Hodgkin''s lymphoma cells. Here, we addressed the question whether deregulated c-FLIP expression in urothelial carcinoma impinges on the ability of death ligands to induce apoptosis. In particular, we investigated the role of the c-FLIP splice variants c-FLIP_long (c-FLIP_L) and c-FLIP_short (c-FLIP_S), which can have opposing functions. We observed diminished expression of the c-FLIP_L isoform in urothelial carcinoma tissues as well as in established carcinoma cell lines compared with normal urothelial tissues and cells, whereas c-FLIP_S was unchanged. Overexpression and RNA interference studies in urothelial cell lines nevertheless demonstrated that c-FLIP remained a crucial factor conferring resistance towards induction of apoptosis by death ligands CD95L and TRAIL. Isoform-specific RNA interference showed c-FLIP_L to be of particular importance. Thus, urothelial carcinoma cells appear to fine-tune c-FLIP expression to a level sufficient for protection against activation of apoptosis by the extrinsic pathway. Therefore, targeting c-FLIP, and especially the c-FLIP_L isoform, may facilitate apoptosis-based therapies of bladder cancer in otherwise resistant tumours.     

c-FLIP Protects Eosinophils from TNF-α-Mediated Cell Death In Vivo

Understanding the signals that regulate eosinophil survival and death is critical to developing new treatments for asthma, atopy, and gastrointestinal disease. Previous studies suggest that TNF-α stimulation protects eosinophils from apoptosis, and this TNF-α-mediated protection is mediated by the upregulation of an unknown protein by NF-κB. Here, we show for the first time that eosinophils express the caspase 8-inhibitory protein c-FLIP, and c-FLIP expression is upregulated upon TNF-α stimulation. Considering that c-FLIP expression is regulated by NF-κB, we hypothesized that c-FLIP might serve as the “molecular switch” that converts TNFRI activation to a pro-survival signal in eosinophils. Indeed, we found that one c-FLIP isoform, c-FLIP_L, is required for mouse eosinophil survival in the presence of TNF-α both in vitro and in vivo. Importantly, our results suggest c-FLIP as a potential therapeutic target for the treatment of eosinophil-mediated disease.     

The C-terminal Domain of the Long Form of Cellular FLICE-inhibitory Protein (c-FLIPL) Inhibits the Interaction of the Caspase 8 Prodomain with the Receptor-interacting Protein 1 (RIP1) Death Domain and Regulates Caspase 8-dependent Nuclear Factor κB (NF-κB) Activation

Caspase 8 plays an essential role in the regulation of apoptotic and non-apoptotic signaling pathways. The long form of cellular FLICE-inhibitory protein (c-FLIP_L) has been shown previously to regulate caspase 8-dependent nuclear factor κB (NF-κB) activation by receptor-interacting protein 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). In this study, the molecular mechanism by which c-FLIP_L regulates caspase 8-dependent NF-κB activation was further explored in the human embryonic kidney cell line HEK 293 and variant cells barely expressing caspase 8. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone greatly diminished caspase 8-dependent NF-κB activation induced by Fas ligand (FasL) when c-FLIP_L, but not its N-terminal fragment c-FLIP(p43), was expressed. The prodomain of caspase 8 was found to interact with the RIP1 death domain and to be sufficient to mediate NF-κB activation induced by FasL or c-FLIP(p43). The interaction of the RIP1 death domain with caspase 8 was inhibited by c-FLIP_L but not c-FLIP(p43). Thus, these results reveal that the C-terminal domain of c-FLIP_L specifically inhibits the interaction of the caspase 8 prodomain with the RIP1 death domain and, thereby, regulates caspase 8-dependent NF-κB activation.     

4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) targets mRNA of the c-FLIP variants and induces apoptosis in MCF-7 human breast cancer cells

Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor for the tumor necrosis factor-related apoptosis-inducing ligand TRAIL and in drug resistance in human malignancies. c-FLIP is an antagonist of caspases-8 and -10, which inhibits apoptosis and is expressed as long (c-FLIP_L) and short (c-FLIP_S) splice forms. c-FLIP is often overexpressed in various human cancers, including breast cancer. Several studies have shown that silencing c-FLIP by specific siRNAs sensitizes cancer cells to TRAIL and anticancer agents. However, systemic use of siRNA as a therapeutic agent is not practical at present. In order to reduce or inhibit c-FLIP expression, small molecules are needed to allow targeting c-FLIP without inhibiting caspases-8 and -10. We used a small molecule inhibitor of c-FLIP, 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), and show that CMH, but not its inactive analog, downregulated c-FLIP_L and c-FLIP_S mRNA and protein levels, caused poly(ADP-ribose) polymerase (PARP) degradation, reduced cell survival, and induced apoptosis in MCF-7 breast cancer cells. These results revealed that c-FLIP is a critical apoptosis regulator that can serve as a target for small molecule inhibitors that downregulate its expression and serve as effective targeted therapeutics against breast cancer cells.     

Cellular FLICE-inhibitory protein (c-FLIP) signalling: A key regulator of receptor-mediated apoptosis in physiologic context and in cancer

Cellular FLICE-inhibitory protein (c-FLIP) is a catalytically inactive procaspase-8/10 homologue that associates with the signalling complex downstream of death-receptors negatively interfering with apoptotic signalling. Three c-FLIP splice variants have been identified: c-FLIP_L, c-FLIP_S and c-FLIP_R, with all three functioning as apoptosis inhibitors involved in modulation of caspase-8/10 activity in both physiologic and pathologic contexts. Furthermore, a cell-type specific pro-apoptotic role, depending on caspase-8 to c-FLIP_L ratio, has also been described for the long isoform. The present review summarizes recent findings concerning c-FLIP proteins’ function and regulation, with a main focus on the c-FLIP_L deregulated expression in cancer. The role of c-FLIP_L as anti-apoptotic pro-survival factor in tumors and the potential utility of this molecule as a possible alternative therapeutic target are discussed.     

The c-FLIPL Cleavage Product p43FLIP Promotes Activation of Extracellular Signal-regulated Kinase (ERK), Nuclear Factor κB (NF-κB), and Caspase-8 and T Cell Survival

Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIP_L can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIP_L. This triggers cleavage of c-FLIP_L at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.     

c-FLIP-Short Reduces Type I Interferon Production and Increases Viremia with Coxsackievirus B3

Cellular FLIP (c-FLIP) is an enzymatically inactive paralogue of caspase-8 and as such can block death receptor-induced apoptosis. However, independent of death receptors, c-FLIP-Long (c-FLIP_L) can heterodimerize with and activate caspase-8. This is critical for promoting the growth and survival of T lymphocytes as well as the regulation of the RIG-I helicase pathway for type I interferon production in response to viral infections. Truncated forms of FLIP also exist in mammalian cells (c-FLIP_S) and certain viruses (v-FLIP), which lack the C-terminal domain that activates caspase-8. Thus, the ratio of c-FLIP_L to these short forms of FLIP may greatly influence the outcome of an immune response. We examined this model in mice transgenically expressing c-FLIP_S in T cells during infection with Coxsackievirus B3 (CVB3). In contrast to our earlier findings of reduced myocarditis and mortality with CVB3 infection of c-FLIP_L-transgenic mice, c-FLIP_S-transgenic mice were highly sensitive to CVB3 infection as manifested by increased cardiac virus titers, myocarditis score, and mortality compared to wild-type C57BL/6 mice. This observation was paralleled by a reduction in serum levels of IL-10 and IFN-α in CVB3-infected c-FLIP_S mice. In vitro infection of c-FLIP_S T cells with CVB3 confirmed these results. Furthermore, molecular studies revealed that following infection of cells with CVB3, c-FLIP_L associates with mitochondrial antiviral signaling protein (MAVS), increases caspase-8 activity and type I IFN production, and reduces viral replication, whereas c-FLIP_S promotes the opposite phenotype.     

Caspase-8, c-FLIP, and caspase-9 in c-Myc-induced apoptosis of fibroblasts

c-Myc is known to induce or potentiate apoptotic processes predominantly by triggering or enhancing the activity of caspases, but the activation mechanisms of caspases by c-Myc remain still poorly understood. Here we found that in MycER™ rat fibroblasts the activation of c-Myc led to an early activation and cleavage of the initiator caspase-8, and concurrent processing and activation of the effector caspases 3 and 7. Interestingly, the expression of cellular FLICE inhibitory protein (c-FLIP) mRNA and the encoded protein, c-FLIP_L, a catalytically inactive homologue of caspase-8, were down-regulated prior to or coincidently with the activation of caspase-8. Of the other known initiators, caspase-9, involved in the mitochondrial pathway, was activated/processed surprisingly late, only after the effector caspases 3/7. Further, we studied the potential involvement of the Fas- and tumor necrosis factor receptor (TNFR)-mediated signaling in the activation of caspase-8 by c-Myc. Blocking of the function of these death receptors by neutralizing antibodies against Fas ligand and TNF-α did not prevent the processing of caspase-8 or cell death. c-Myc was neither found to induce any changes in the expression of TNF-related apoptosis inducing ligand (TRAIL) or its receptor. These data suggest that caspase-8 does not become activated through an extrinsic but an “intrinsic/intracellular” apoptotic pathway unleashed by the down-regulation of c-FLIP by c-Myc. Moreover, ectopic expression of c-FLIP_L inhibited the c-Myc-induced apoptosis.     

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation

Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5^−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5^−/− or Atg7^−/− MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5^−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation.     

Caspase-8 activity has an essential role in CD95/Fas-mediated MAPK activation

Stimulation of CD95/Fas/APO-1 results in the induction of both apoptotic and non-apoptotic signaling pathways. The processes regulating these two opposing pathways have not been thoroughly elucidated to date. In this study, using quantitative immunoblots, imaging, and mathematical modeling, we addressed the dynamics of the DED proteins of the death-inducing signaling complex (DISC), procaspase-8, and cellular FLICE inhibitory proteins (c-FLIPs) to the onset of CD95-mediated ERK1/2 and p38 mitogen-activated protein kinase (MAPK) activation. We found that CD95 DISC-induced caspase-8 activity is important for the initiation of ERK1/2 and p38 MAPK activation. The long c-FLIP isoform, c-FLIP_L, and the short c-FLIP isoform, c-FLIP_R, inhibited MAPK induction by blocking caspase-8 processing at the DISC. Furthermore, we built a mathematical model describing CD95 DISC-mediated MAPK activation and apoptosis. The model quantitatively defined the dynamics of DED proteins, procaspase-8, and c-FLIP, which lead to caspase-8 activation and induction of apoptotic and non-apoptotic signaling pathways. In conclusion, the combination of biochemical analysis with mathematical modeling provides evidence for an important role of caspase-8 in CD95-mediated activation of MAPKs, while c-FLIP exerts a regulatory function in this process.     

The endogenous caspase-8 inhibitor c-FLIPL regulates ER morphology and crosstalk with mitochondria

Components of the death receptor-mediated pathways like caspase-8 have been identified in complexes at intracellular membranes to spatially restrict the processing of local targets. In this study, we report that the long isoform of the cellular FLICE-inhibitory protein (c-FLIP_L), a well-known inhibitor of the extrinsic cell death initiator caspase-8, localizes at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs). ER morphology was disrupted and ER Ca²⁺-release as well as ER-mitochondria tethering was decreased in c-FLIP^−/− mouse embryonic fibroblasts (MEFs). Mechanistically, c-FLIP ablation resulted in enhanced basal caspase-8 activation and in caspase-mediated processing of the ER-shaping protein reticulon-4 (RTN4) that was corrected by re-introduction of c-FLIP_L and caspase inhibition, resulting in the recovery of a normal ER morphology and ER-mitochondria juxtaposition. Thus, the caspase-8 inhibitor c-FLIP_L emerges as a component of the MAMs signaling platforms, where caspases appear to regulate ER morphology and ER-mitochondria crosstalk by impinging on ER-shaping proteins like the RTN4.Cellular FLICE inhibitory proteins (c-FLIP) inhibit death receptor (DR)-mediated apoptosis, by preventing caspase-8 activation.¹ Among the three identified c-FLIP splicing forms,^{2, 3} c-FLIP_S,R were described as cytosolic, whereas c-FLIP_L was also observed in the nucleus. A pool of membrane-bound c-FLIP_L was also described⁴ suggesting that caspase-8/c-FLIP_L could re-distribute on stimulation, leading to a more subtle regulation of caspase-8 activity depending on substrates localization.⁵ Furthermore, caspase-8 itself and Fas-Associated Death Domain adaptor protein (FADD) were found or were shown to re-loca5lize in local complexes on ER^{6, 7, 8} and mitochondria,^{9, 10} mediating the exchange of signals between the two organelles.^{11, 12, 13} Several molecular platforms containing both membrane-bound proteins and cytosolic apoptosis modulators have been identified at the ER-mitochondria interface (the so-called mitochondria-associated membranes or MAMs),¹⁴ controlling ER-mitochondria anchorage as well as lipid metabolism, Ca²⁺ signaling and apoptosis.¹⁵ MAMs have been recently described as lipid raft-like domains that orient proteins to promote the ER-mitochondria juxtaposition;¹⁶ consequently, alterations in their composition may profoundly affect the physical and functional inter-organelle crosstalk. Furthermore, as mitochondrial and ER membranes are continuously and concertedly remodeled,¹⁷ it is not surprising that membrane-shaping proteins can also exert a function in regulating the ER-mitochondria coupling.^{12, 18} Different families of ER-shaping proteins control the organization of peripheral ER, which consists of sheet-like cisternae and tubules connected by three-way junctions.¹⁹ Among these, Reticulons (RTN) and Deleted in Polyposis locus 1 (DP1) proteins cause the ER membrane to curve and tubulate,^{20, 21} whereas the GTPases Atlastins (ATL) promote the branching of ER tubules;²² finally, ER sheet-enriched proteins such as the 63-kDa cytoskeleton-linking membrane protein (CLIMP63) control the width of ER cisternae, anchoring the organelle to microtubules and maintaining its spatial distribution.^{23, 24} Along with other components of the extrinsic apoptosis, here we described for the first time the enrichment of c-FLIP_L at ER and ER-mitochondria interface. Furthermore, we observed that ER structure and tethering to mitochondria are impaired in cells lacking c-FLIP. Given the importance of membrane-shaping proteins and MAM complexes in regulating organelles structure and ER-mitochondria juxtaposition, we focused on the mechanism underlying this phenotype and we found that c-FLIP_L deficiency induces the caspase-mediated processing of RTN4, thus affecting organelle shape and coupling to mitochondria. We therefore concluded that c-FLIP_L is a novel regulator of ER morphology and ER-mitochondria crosstalk.     

killerFLIP: a novel lytic peptide specifically inducing cancer cell death

One of the objectives in the development of effective cancer therapy is induction of tumor-selective cell death. Toward this end, we have identified a small peptide that, when introduced into cells via a TAT cell-delivery system, shows a remarkably potent cytoxicity in a variety of cancer cell lines and inhibits tumor growth in vivo, whereas sparing normal cells and tissues. This fusion peptide was named killerFLIP as its sequence was derived from the C-terminal domain of c-FLIP, an anti-apoptotic protein. Using structure activity analysis, we determined the minimal bioactive core of killerFLIP, namely killerFLIP-E. Structural analysis of cells using electron microscopy demonstrated that killerFLIP-E triggers cell death accompanied by rapid (within minutes) plasma membrane permeabilization. Studies of the structure of the active core of killerFLIP (-E) indicated that it possesses amphiphilic properties and self-assembles into micellar structures in aqueous solution. The biochemical properties of killerFLIP are comparable to those of cationic lytic peptides, which participate in defense against pathogens and have also demonstrated anticancer properties. We show that the pro-cell death effects of killerFLIP are independent of its sequence similarity with c-FLIP_L as killerFLIP-induced cell death was largely apoptosis and necroptosis independent. A killerFLIP-E variant containing a scrambled c-FLIP_L motif indeed induced similar cell death, suggesting the importance of the c-FLIP_L residues but not of their sequence. Thus, we report the discovery of a promising synthetic peptide with novel anticancer activity in vitro and in vivo.     

ARG2 impairs endothelial autophagy through regulation of MTOR and PRKAA/AMPK signaling in advanced atherosclerosis

Impaired autophagy function and enhanced ARG2 (arginase 2)-MTOR (mechanistic target of rapamycin) crosstalk are implicated in vascular aging and atherosclerosis. We are interested in the role of ARG2 and the potential underlying mechanism(s) in modulation of endothelial autophagy. Using human nonsenescent “young” and replicative senescent endothelial cells as well as Apolipoprotein E-deficient (apoe^−/−Arg2^+/+) and Arg2-deficient apoe^−/− (apoe^−/−arg2^−/−) mice fed a high-fat diet for 10 wk as the atherosclerotic animal model, we show here that overexpression of ARG2 in the young cells suppresses endothelial autophagy with concomitant enhanced expression of RICTOR, the essential component of the MTORC2 complex, leading to activation of the AKT-MTORC1-RPS6KB1/S6K1 (ribosomal protein S6 kinase, 70kDa, polypeptide 1) cascade and inhibition of PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit). Expression of an inactive ARG2 mutant (H160F) had the same effect. Moreover, silencing RPS6KB1 or expression of a constitutively active PRKAA prevented autophagy suppression by ARG2 or H160F. In senescent cells, enhanced ARG2-RICTOR-AKT-MTORC1-RPS6KB1 and decreased PRKAA signaling and autophagy were observed, which was reversed by silencing ARG2 but not by arginase inhibitors. In line with the above observations, genetic ablation of Arg2 in apoe^−/− mice reduced RPS6KB1, enhanced PRKAA signaling and endothelial autophagy in aortas, which was associated with reduced atherosclerosis lesion formation. Taken together, the results demonstrate that ARG2 impairs endothelial autophagy independently of the L-arginine ureahydrolase activity through activation of RPS6KB1 and inhibition of PRKAA, which is implicated in atherogenesis.     

Tribbles homolog 3-mediated targeting the AKT/mTOR axis in mice with retinal degeneration

Various retinal degenerative disorders manifest in alterations of the AKT/mTOR axis. Despite this, consensus on the therapeutic targeting of mTOR in degenerating retinas has not yet been achieved. Therefore, we investigated the role of AKT/mTOR signaling in rd16 retinas, in which we restored the AKT/mTOR axis by genetic ablation of pseudokinase TRB3, known to inhibit phosphorylation of AKT and mTOR. First, we found that TRB3 ablation resulted in preservation of photoreceptor function in degenerating retinas. Then, we learned that the mTOR downstream cellular pathways involved in the homeostasis of photoreceptors were also reprogrammed in rd16 TRB3^−/− retinas. Thus, the level of inactivated translational repressor p-4E-BP1 was significantly increased in these mice along with the restoration of translational rate. Moreover, in rd16 mice manifesting decline in p-mTOR at P15, we found elevated expression of Beclin-1 and ATG5 autophagy genes. Thus, these mice showed impaired autophagy flux measured as an increase in LC3 conversion and p62 accumulation. In addition, the RFP-EGFP-LC3 transgene expression in rd16 retinas resulted in statistically fewer numbers of red puncta in photoreceptors, suggesting impaired late autophagic vacuoles. In contrast, TRIB3 ablation in these mice resulted in improved autophagy flux. The restoration of translation rate and the boost in autophagosome formation occurred concomitantly with an increase in total Ub and rhodopsin protein levels and the elevation of E3 ligase Parkin1. We propose that TRB3 may retard retinal degeneration and be a promising therapeutic target to treat various retinal degenerative disorders.Subject terms: Translation, Diseases     

The α1D-adrenergic receptor is expressed intracellularly and coupled to increases in intracellular calcium and reactive oxygen species in human aortic smooth muscle cells

Background

We have previously shown that c-FLIP_L is a more potent inhibitor than c-FLIP_S of Fas ligand-induced apoptosis and that c-FLIP_L physically binds to Daxx, an alternative Fas-signaling adaptor. Here we examined whether c-FLIP_S effectively inhibits TNFR1-mediated apoptosis and triggers JNK activation through its interaction with TRAF2.

Results

Some cancer cell lines, such as DU145, AGS, and PC3, have higher levels of c-FLIP_S than other cell lines, such as SNU-719 and T24. The expression of c-FLIP_S correlated with the susceptibility to TNFR1-mediated apoptosis. In contrast to DU145 and PC3, which are resistant to TNFR1-mediated apoptosis, T24 and SNU719 were sensitive to TNF-α treatment. To address the role of c-FLIP_S in TNFR1-mediated apoptosis, we examined the molecular interaction between c-FLIP_S and TRAF2. As expected, western blot analysis revealed that TRAF2 antibody immunoprecipitated a greater amount of c-FLIP_S than c-FLIP_L. Also, we measured the involvement of c-FLIP_S in TNF-α-induced JNK activation and apoptosis by comparing these in TNF-α-resistant and TNF-α-sensitive cell lines. Treatment with TNF-α increased the phosphorylated JNK level in SNU719 and T24 cells, whereas DU145 and AGS cells were resistant to TNF-α-mediated apoptosis.

Conclusion

We now report that the short form of c-FLIP_S is a more efficient inhibitor of TNF-receptor 1-mediated apoptosis signaling than the long form of the protein.