c-FLIP Isoforms Research Articles

CFLIPS regulates alternative NLRP3 inflammasome activation in human monocytes.

The innate immune responses, including inflammasome activation, are paramount for host defense against pathogen infection. In contrast to canonical and noncanonical inflammasome activation, in this study, heat-killed gram-negative bacteria (HK bacteria) were identified as single-step stimulators of the NLRP3 inflammasome in human monocytes, and they caused a moderate amount of IL-1β to be released from cells. Time course experiments showed that this alternative inflammasome response was finished within a few hours. Further analysis showed that the intrinsically limited NLRP3 inflammasome activation response was due to the negative regulation of caspase-8 by the short isoform of cFLIP (cFLIPs), which was activated by NF-κB. In contrast, overexpressed cFLIPS, but not overexpressed cFLIPL, inhibited the activation of caspase-8 and the release of IL-1β in response to HK bacteria infection in human monocytes. Furthermore, we demonstrated that TAK1 activity mediated the expression of cFLIPs and was upstream and essential for the caspase-8 cleavage induced by HK bacteria in human monocytes. The functional specificity of cFLIPs and TAK1 revealed unique responses of human monocytes to a noninvasive pathogen, providing novel insights into an alternative regulatory pathway of NLRP3 inflammasome activation.

CRISPR screens identify novel regulators of cFLIP dependency and ligand-independent, TRAIL-R1-mediated cell death.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). PEL cell lines require expression of the cellular FLICE inhibitory protein (cFLIP) for survival, although KSHV encodes a viral homolog of this protein (vFLIP). Cellular and viral FLIP proteins have several functions, including, most importantly, the inhibition of pro-apoptotic caspase 8 and modulation of NF-κB signaling. To investigate the essential role of cFLIP and its potential redundancy with vFLIP in PEL cells, we first performed rescue experiments with human or viral FLIP proteins known to affect FLIP target pathways differently. The long and short isoforms of cFLIP and molluscum contagiosum virus MC159L, which are all strong caspase 8 inhibitors, efficiently rescued the loss of endogenous cFLIP activity in PEL cells. KSHV vFLIP was unable to fully rescue the loss of endogenous cFLIP and is therefore functionally distinct. Next, we employed genome-wide CRISPR/Cas9 synthetic rescue screens to identify loss of function perturbations that can compensate for cFLIP knockout. Results from these screens and our validation experiments implicate the canonical cFLIP target caspase 8 and TRAIL receptor 1 (TRAIL-R1 or TNFRSF10A) in promoting constitutive death signaling in PEL cells. However, this process was independent of TRAIL receptor 2 or TRAIL, the latter of which is not detectable in PEL cell cultures. The requirement for cFLIP is also overcome by inactivation of the ER/Golgi resident chondroitin sulfate proteoglycan synthesis and UFMylation pathways, Jagunal homolog 1 (JAGN1) or CXCR4. UFMylation and JAGN1, but not chondroitin sulfate proteoglycan synthesis or CXCR4, contribute to TRAIL-R1 expression. In sum, our work shows that cFLIP is required in PEL cells to inhibit ligand-independent TRAIL-R1 cell death signaling downstream of a complex set of ER/Golgi-associated processes that have not previously been implicated in cFLIP or TRAIL-R1 function.

An engineered construct of cFLIP provides insight into DED1 structure and interactions

Cellular FLICE-like inhibitory protein (cFLIP) is a member of the Death Domain superfamily with pivotal roles in many cellular processes and disease states, including cancer and autoimmune disorders. In the context of the death-inducing signaling complex (DISC), cFLIP isoforms regulate extrinsic apoptosis by controlling procaspase-8 activation. The function of cFLIP is mediated through a series of protein-protein interactions, engaging the two N-terminal death effector domains (DEDs). Here, we solve the structure of an engineered DED1 domain of cFLIP using solution nuclear magnetic resonance (NMR) and we define the interaction with FADD and calmodulin, protein-protein interactions that regulate the function of cFLIP in the DISC. cFLIP DED1 assumes a canonical DED fold characterized by six α helices and is able to bind calmodulin and FADD through two separate interfaces. Our results clearly demonstrate the role of DED1 in the cFLIP/FADD association and contribute to the understanding of the assembly of DISC filaments.

Cellular FADD-like IL-1β-converting enzyme-inhibitory protein attenuates myocardial ischemia/reperfusion injury via suppressing apoptosis and autophagy simultaneously

Background and aimsMyocardial ischemia/reperfusion injury (MI/RI) is a result of coronary revascularization, and often increases cell apoptosis and autophagy. Downregulated cellular FADD-like-IL-1β-converting enzyme-inhibitory protein (cFLIP) was associated with development of several myocardial diseases, whether overexpression of cFLIP can attenuate MI/RI remains unclear. This study aimed to determine the effects of cFLIP on apoptosis and autophagy in MI/RI. Methods and resultsIschemia/reperfusion (I/R) rat model and hypoxia/reoxygenation (H/R) cardiomyocytes model were established. Both I/R injury and H/R injury down-regulated expression of two cFLIP isoforms (cFLIPL and cFLIPS), and instigated apoptosis and autophagy simultaneously. Overexpression of cFLIPL and/or cFLIPS led to a significant increase in cardiomyocytes viability in vitro, and also reduced the myocardial infarct volume in vivo, these changes were associated with suppressed apoptosis and autophagy. Mechanistically, overexpression of cFLIP significantly downregulated pro-apoptotic molecules (Caspase-3, -8, -9), and pro-autophagic molecules (Beclin-1 and LC3-II). Moreover, cFLIP significantly suppressed activity of NF-κB pathway to upregulate the expression of Bcl-2, which is the molecular of interplay of apoptosis and autophagy. ConclusionOverexpression of cFLIP significantly attenuated MI/RI both in vivo and vitro via suppression of apoptosis and lethal autophagy. cFLIP can suppress activity of NF-κB pathway, and further upregulated expression of Bcl-2.

Activated human NK and CD8+ T cells express both TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL receptors but are resistant to TRAIL-mediated cytotoxicity

AbstractThe expression of tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) and TRAIL receptors was investigated in resting and cytokine-activated purified primary human natural killer (NK) and CD8+ T cells. Resting NK and CD8+ T cells expressed the mRNA for all TRAIL receptors, but TRAIL-R4 was the only receptor clearly detectable on the surface of both cell types. NK cells were activated by interleukin 2 (IL-2) or IL-15, whereas CD8+ T cells were activated by phytohemagglutinin (PHA) + IL-2 followed by IL-2 alone for up to 10 days. On activation, both cell types rapidly expressed TRAIL-R2 and TRAIL-R3, whose expression peaked at day 10 of culture. TRAIL-R1, however, was never expressed at any time point examined, whereas the expression of TRAIL-R4, which showed a progressive increase in CD8+ T cells, remained constant in NK cells. Notwithstanding the expression of TRAIL-R2, recombinant TRAIL did not show any cytotoxic activity on either NK or CD8+ T cells. Both resting and activated NK and CD8+ T cells were found to express high levels of the 2 isoforms of c-FLIP (cellular Fas-associated death domain protein [FADD]–like IL-1–converting enzyme [FLICE]–inhibitory protein). Small interference RNA-mediated inhibition of c-FLIP expression in NK cells abrogated their resistance to the apoptotic effect of soluble TRAIL. Thus, once activated the major cytotoxic effector cells are potentially sensitive to TRAIL but are physiologically protected from its apoptotic action by intracellular level of c-FLIP.

CFLIPL protects macrophages from LPS-induced pyroptosis via inhibition of complex II formation.

Cell death and inflammation are interdependent host responses to infection. During pyroptotic cell death, interleukin-1β (IL-1β) release occurs through caspase-1 and caspase-11-mediated gasdermin D pore formation. In vivo, responses to lipopolysaccharide (LPS) result in IL-1β secretion. In vitro, however, murine macrophages require a second "danger signal" for the inflammasome-driven maturation of IL-1β. Recent reports have shown caspase-8-mediated pyroptosis in LPS-activated macrophages but have provided conflicting evidence regarding the release of IL-1β under these conditions. Here, to further characterize the mechanism of LPS-induced secretion in vitro, we reveal an important role for cellular FLICE-like inhibitory protein (cFLIP) in the regulation of the inflammatory response. Specifically, we show that deficiency of the long isoform cFLIPL promotes complex II formation, driving pyroptosis, and the secretion of IL-1β in response to LPS alone.

Macrophages cFLIP out

Pyroptosis Pathogens have evolved to survive within hosts in part by interfering with host signaling cascades. Yersinia bacteria use the effector protein YopJ to thwart MAP kinase signaling downstream of Toll-like receptor activation. In response to YopJ, host cells can release interleukin-1β and initiate pyroptosis by inhibition of the kinase TAK1 and subsequent caspase-8–directed cleavage of gasdermin D, a protein that forms cell membrane pores. Muendlein et al. report that cFLIP, a major antiapoptotic regulator, plays a central role in this process. Knockdowns of the long but not short cFLIP isoform in macrophages removes the requirement for TAK1 inhibition. Rather, deficiency of the long isoform fuels caspase-8 activation, mitochondrial complex II formation, pyroptosis, and interleukin-1β secretion in response to lipopolysaccharide alone, underscoring its importance for cell death and inflammation. Science , this issue p. [1379][1] [1]: /lookup/doi/10.1126/science.aay3878

Long and short isoforms of c-FLIP act as control checkpoints of DED filament assembly.

The assembly of the death-inducing signaling complex (DISC) and death effector domain (DED) filaments at CD95/Fas initiates extrinsic apoptosis. Procaspase-8 activation at the DED filaments is controlled by short and long c-FLIP isoforms. Despite apparent progress in understanding the assembly of CD95-activated platforms and DED filaments, the detailed molecular mechanism of c-FLIP action remains elusive. Here, we further addressed the mechanisms of c-FLIP action at the DISC using biochemical assays, quantitative mass spectrometry, and structural modeling. Our data strongly indicate that c-FLIP can bind to both FADD and procaspase-8 at the DED filament. Moreover, the constructed in silico model shows that c-FLIP proteins can lead to the formation of the DISCs comprising short DED filaments as well as serve as bridging motifs for building a cooperative DISC network, in which adjacent CD95 DISCs are connected by DED filaments. This network is based on selective interactions of FADD with both c-FLIP and procaspase-8. Hence, c-FLIP proteins at the DISC control initiation, elongation, and composition of DED filaments, playing the role of control checkpoints. These findings provide new insights into DISC and DED filament regulation and open innovative possibilities for targeting the extrinsic apoptosis pathway.

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.

Differential expression patterns of the short and long isoform of cFLIP on FasL‑mediated apoptosis.

cFLIP is a key regulator of the anti‑apoptotic mechanism and its association with FAS‑mediated apoptosis has been widely studied and well documented. However, the equipoise between its two isoforms i.e. the long isoform cFLIP(L) and the short isoform cFLIP(S) during FAS‑mediated apoptosis remains to be revealed. Therefore, the present study aimed to investigate the regulatory effect of these isoforms on FasL‑mediated apoptosis in renal carcinoma. Our results revealed that FasL treatment to Caki cells induced the expression of cFLIP(S) and downregulated the expression of cFLIP(L) in a concentration‑ and time‑dependent manner. Furthermore, our results indicated that cell death receptor‑mediated apoptosis inducers such as TNF‑α and TRAIL, induced apoptosis in Caki cells along with downregulation of cFLIP(L), however, they had no effect on the expression of cFLIP(S). In addition, FasL‑mediated cFLIP(L) downregulation was prevented by pan‑caspase inhibitor (z‑VAD‑fmk), however pan‑caspase inhibitor did not have an effect on FasL‑induced cFLIP(S) upregulation. Furthermore, FasL induced upregulation of the expression of cFLIP(S) at the post‑translational level. Furthermore, pretreatment of Caki cells with ROS scavengers (N‑acetylcysteine and glutathione) prevented the downregulation of cFLIP(L), the upregulation of cFLIP(S) and apoptosis induced by FasL. Collectively, these data indicated that a novel pathway of cFLIP(L)/(S) differential expression pattern was associated with FasL‑induced apoptosis and modulated by ROS generation.

Expression of c-FLIP in a rat model of sepsis and its effects on endothelial apoptosis.

Sepsis is characterized by the impaired regulation of inflammatory responses. Apoptosis is important in the pathogenesis of sepsis. Cellular FLICE-inhibitory protein (c-FLIP) is a catalytically inactive caspase-8 homologue, which negatively interferes with apoptotic signaling. The role of c-FLIP in sepsis and in endothelial cell apoptosis, a critical step in the pathogenesis of sepsis, remains controversial. In the present study, to investigate the relationship between c-FLIP and sepsis, a rat model of sepsis was induced by cecal ligation and puncture, and western blot analysis was used to detect the expression of c-FLIPL, the long isoform of c-FLIP. Lower protein expression levels of c-FLIPL were found in the brain, intestine and lung of the rat sepsis model, compared with the rats in the sham surgery group. The association between the expression of c-FLIPL and endothelial cell apoptosis was further examined in vitro by c-FLIPL overexpression and flow cytometry, which demonstrated that the expression of c-FLIPL was inversely correlated with endothelial cell apoptosis. These data suggested that c-FLIP may be important in sepsis and shed light on therapeutic strategies.

Co-operative and Hierarchical Binding of c-FLIP and Caspase-8: A Unified Model Defines How c-FLIP Isoforms Differentially Control Cell Fate

SummaryThe death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIPL only) or anti-apoptotic (c-FLIPL/c-FLIPS) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIPL/S binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate.

Balance between short and long isoforms of cFLIP regulates Fas-mediated apoptosis in vivo

cFLIP, an inhibitor of apoptosis, is a crucial regulator of cellular death by apoptosis and necroptosis; its importance in development is exemplified by the embryonic lethality in cFLIP-deficient animals. A homolog of caspase 8 (CASP8), cFLIP exists in two main isoforms: cFLIPL (long) and cFLIPR (short). Although both splice variants regulate death receptor (DR)-induced apoptosis by CASP8, the specific role of each isoform is poorly understood. Here, we report a previously unidentified model of resistance to Fas receptor-mediated liver failure in the wild-derived MSM strain, compared with susceptibility in C57BL/6 (B6) mice. Linkage analysis in F2 intercross (B6 x MSM) progeny identified several MSM loci controlling resistance to Fas-mediated death, including the caspase 8- and FADD-like apoptosis regulator (Cflar) locus encoding cFLIP. Furthermore, we identified a 21-bp insertion in the 3' UTR of the fifth exon of Cflar in MSM that influences differential splicing of cFLIP mRNA. Intriguingly, we observed that MSM liver cells predominantly express the FLIPL variant, in contrast to B6 liver cells, which have higher levels of cFLIPR. In keeping with this finding, genome-wide RNA sequencing revealed a relative abundance of FLIPL transcripts in MSM hepatocytes whereas B6 liver cells had significantly more FLIPR mRNA. Importantly, we show that, in the MSM liver, CASP8 is present exclusively as its cleaved p43 product, bound to cFLIPL. Because of partial enzymatic activity of the heterodimer, it might prevent necroptosis. On the other hand, it prevents cleavage of CASP8 to p10/20 necessary for cleavage of caspase 3 and, thus, apoptosis induction. Therefore, MSM hepatocytes are predisposed for protection from DR-mediated cell death.

Estradiol Upregulates c-FLIPlong Expression in Anterior Pituitary Cells

Anterior pituitary cell turnover depends on a tight balance between proliferation and apoptosis. We have previously shown that estrogens sensitize anterior pituitary cells to pro-apoptotic stimuli. c-FLIP (cellular-FLICE-inhibitory-protein) isoforms are regulatory proteins of apoptosis triggered by death receptors. c-FLIPshort isoform competes with procaspase-8 inhibiting its activation. However, c-FLIPlong isoform may have a pro- or anti-apoptotic function depending on its expression level. In the present study, we explored whether estrogens modulate c-FLIP expression in anterior pituitary cells from ovariectomized (OVX) rats and in GH3 cells, a somatolactotrope cell line. Acute administration of 17β-estradiol to OVX rats increased c-FLIPlong expression in the anterior pituitary gland without changing c-FLIPshort expression as assessed by Western blot. Estradiol in vitro also increased c-FLIPlong expression in anterior pituitary cells but not in GH3 cells. As determined by flow cytometry, the percentage of anterior pituitary cells expressing c-FLIP was higher than in GH3 cells. However, c-FLIP fluorescence intensity in GH3 cells was higher than in anterior pituitary cells. FasL increased the percentage of TUNEL-positive GH3 cells incubated either with or without estradiol suggesting that the pro-apoptotic action of Fas activation is estrogen-independent. Our results show that unlike what happens in nontumoral pituitary cells, estrogens do not modulate either c-FLIPlong expression or FasL-induced apoptosis in GH3 cells. The stimulatory effect of estradiol on c-FLIPlong expression could be involved in the sensitizing effect of this steroid to apoptosis in anterior pituitary cells. The absence of this estrogenic action in tumor pituitary cells could be involved in their tumor-like behavior.

Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain.

The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, procaspase-8, procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and procaspase-10 that are present in 10 times lower amounts compared with procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.

Expression and localization of cFLIP anti-apoptotic protein in the porcine corpus luteum and corpora albicans during the estrous cycle and pregnancy.

We determined expression and localization of the anti-apoptotic cellular FLICE inhibitory protein (cFLIP) in the porcine corpora lutea (CL) and corpus albicans (CA) during estrous and pregnancy. The CL and CA were collected at different stages of estrous to determine cFLIP immunolocalization, and mRNA and protein expression. The mRNA expression of the short cFLIP isoform (cFLIPS) was higher at the early and mid CL stages, and lower by the late CL stage (P < 0.01); mRNA expression of the long cFLIP isoform (cFLIPL) was higher at the mid CL stage, and lower at the early and late CL stages (P < 0.01). Levels of cFLIPS and cFLIPL were steady and high during the early and mid CL stages, and had significantly decreased (P < 0.01) by the late stage. The cFLIP protein was highly expressed in the early and mid CL stages of estrous, but weakly ex-pressed in the late stage. Expression of cFLIPS showed no significant difference between preovulatory corpus albicans (CA1) and corpus albicans (CA2) coexistent with the CL from the previous estrus, but cFLIPL mRNA expression was higher during CA1 than CA2. The expression of cFLIPS showed no significant difference between CA1 and CA2, but cFLIPL was not detected. The cFLIP protein was weak-ly expressed in the CA. Expression of cFLIPS and cFLIPL mRNA and proteins was observed in the CL, and the cFLIP protein was highly expressed during pregnancy. We propose that cFLIPS/L acts as a survival factor, and performs an anti-apoptotic function in the porcine CL.

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-FLIPL, 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.

Abstract 2278: The SMAC-mimetic birinapant regulates autocrine TNF production by caspase-8:RIPK1 complex via p38MAPK pathway

Abstract Birinapant (TL32711), a SMAC-mimetic currently in clinical trials, antagonizes multiple members of the inhibitor of apoptosis (IAP) protein family to promote apoptosis in cancer cells through the formation of a caspase-8:RIPK1 complex and induction of autocrine tumor necrosis factor (TNF). Evidently, birinapant-resistant cells do not produce TNF upon drug treatment. However, the mechanism by which birinapant-sensitive cells produce TNF is not clear. Owing to the fact that mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes, we explored the role of MAPKs in birinapant-induced TNF production. Western blot analyses on a panel of birinapant-sensitive (SK-OV-3, EVSA-T, TOV-21G: all with an IC50 &amp;lt; 200nM) and birinapant-resistant (IGROV-1, HCT116, WIDR &amp; SK-MEL-28: all with an IC50 &amp;gt; 10μM) cell lines showed that birinapant induced rapid and robust phosphorylation of p38MAPK in sensitive cells, which corresponded to increased TNF production as assessed by ELISA. However, resistant cells were not affected at multiple time points. The p38MAPK inhibitor, LY2228820, reduced the levels of birinapant-induced TNF (2-fold with 5nM birinapant after 24h) in birinapant-sensitive cancer cells (SK-OV-3), whereas inhibitors of Erk1/2 (PD98059) and JNK (SP600125) had no effect on TNF production. Interestingly, RNAi-mediated inhibition of p38α (MAPK14) isoform significantly inhibited birinapant-induced TNF production (∼3-fold with 5nM birinapant after 24h) in SK-OV-3 cells while inhibition of p38β (MAPK11) isoform upregulated TNF production (∼3-fold), suggesting that there may be a balance between p38MAPK isoforms to regulate TNF production. Pharmacological and RNAi-mediated inhibition of RIPK1 and caspase-8 activities in SK-OV-3 cells resulted in reduced birinapant-induced p38MAPK phosphorylation and TNF production. Furthermore, commercially available pooled siRNAs targeting different isoforms of cFLIP, a catalytically-inactive homologue of caspase-8, upregulated p38MAPK phosphorylation (∼4-fold) and TNF production (∼8 to 26-fold) in birinapant-resistant cancer cell lines (T24, HT1376 &amp; IGROV-1), and conferred differential sensitivity to the drug treatment (T24-IC50: 7nM; HT1376-IC50: &amp;gt;10nM; IGROV-1-IC50: &amp;gt;1μM). Experiments involving cFLIP long (cFLIP-L) and short (cFLIP-S) isoform-specific siRNAs showed that inhibition of cFLIP-L, but not cFLIP-S, in T24 &amp; HT1376 cells induced p38MAPK phosphorylation and TNF production, and conferred sensitivity to birinapant. However, the combined inhibition of cFLIP-L and cFLIP-S in IGROV-1 cells was required to induce p38MAPK phosphorylation, TNF production and apoptosis. Collectively, these observations indicate that birinapant-induced autocrine TNF production is triggered by the activation of the caspase-8/RIPK1/p38MAPK axis, and that cFLIP isoforms may confer a context-dependent negative regulatory effect. Citation Format: Gurpreet Singh Kapoor, Christopher A. Benetatos, Yasuhiro Mitsuuchi, Eric M. Neiman, Guangyao Yu, Mark A. Mckinlay, Jennifer Burns, John Silke, Stephen M. Condon, Srinivas K. Chunduru. The SMAC-mimetic birinapant regulates autocrine TNF production by caspase-8:RIPK1 complex via p38MAPK pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2278. doi:10.1158/1538-7445.AM2014-2278

The regulation and role of c-FLIP in human Th cell differentiation.

The early differentiation of T helper (Th) cells is a tightly controlled and finely balanced process, which involves several factors including cytokines, transcription factors and co-stimulatory molecules. Recent studies have shown that in addition to the regulation of apoptosis, caspase activity is also needed for Th cell proliferation and activation and it might play a role in Th cell differentiation. The isoforms of the cellular FLICE inhibitory protein (c-FLIP) are regulators of CASPASE-8 activity and the short isoform, c-FLIPS, has been shown to be up-regulated by IL-4, the Th2 driving cytokine. In this work, we have studied the expression and functional role of three c-FLIP isoforms during the early Th cell differentiation. Only two of the isoforms, c-FLIPS and c-FLIPL, were detected at the protein level although c-FLIPR was expressed at the mRNA level. The knockdown of c-FLIPL led to enhanced Th1 differentiation and elevated IL-4 production by Th2 cells, whereas the knockdown of c-FLIPS diminished GATA3 expression and IL-4 production by Th2 cells. In summary, our results provide new insight into the role of c-FLIP proteins in the early differentiation of human Th cells.

Class I-specific histone deacetylase inhibitor MS-275 overrides TRAIL-resistance in melanoma cells by downregulating c-FLIP.

Tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) has selective killing effect toward malignant cells; however some human melanomas are intrinsically resistant. In this study, we have shown that class I-specific histone deacetylase inhibitor (HDACi) MS-275 can synergize with TRAIL to induce apoptosis in TRAIL-resistant cell lines and to enhance susceptibility of sensitive cells. Conversely, class II-selective HDACi MC1575 has shown no effect on the resistance of melanoma cells and was able exclusively to increase TRAIL-induced cell death in responsive cells. Both the HDACis variably increased DR4, DR5, and procaspase 8 expression, regardless whether cells were TRAIL-sensitive or TRAIL-resistant. However, only MS-275 markedly decreased the expression levels of both the long and short c-FLIP isoforms. RNAi-mediated c-FLIP silencing resulted in caspase 8-dependent apoptosis in survivor cells which was comparable to that observed following MS-275 treatment. Accordingly, enforced expression of ectopic c-FLIP has abolished the cooperative induction of apoptosis by the combination of MS-275 and TRAIL. These data indicate that c-FLIP is a critical regulator of death ligand sensitivity in melanoma. Inhibition of class I HDAC isoenzymes 1, 2 and 3 has resulted to be functionally important for c-FLIP downregulation by MS-275. In contrast, knockdown of class II HDACs has had no effect on c-FLIP expression, thus explaining the dual incapacity of MC1575 to inhibit c-FLIP expression and sensitize cells resistant to TRAIL. The data reported here suggest that MS-275 represents a promising therapeutic approach in combination with TRAIL for treatment of cutaneous and uveal melanoma due to its ability to reduce c-FLIP expression.