* 603599
CASP8- AND FADD-LIKE APOPTOSIS REGULATOR; CFLAR
Alternative titles; symbols
FLICE INHIBITORY PROTEIN; FLIP
INHIBITOR OF FLICE; I-FLICE
CASPASE-EIGHT-RELATED PROTEIN; CASPER
FADD-LIKE ANTIAPOPTOTIC MOLECULE 1; FLAME1
CASPASE HOMOLOG; CASH
CASPASE-LIKE APOPTOSIS REGULATORY PROTEIN; CLARP
MACH-RELATED INDUCER OF TOXICITY; MRIT
HGNC Approved Gene Symbol: CFLAR
Cytogenetic location: 2q33.1 Genomic coordinates (GRCh38) : 2:201,116,164-201,176,687 (from NCBI)
TEXT
Description
CFLAR is a caspase-8 (CASP8; 601763) inhibitor and plays a role in CASP8-mediated apoptosis (Mora-Molina et al., 2022; Dold et al., 2022).
Cloning and Expression
Caspases are cysteine proteases that play a central role in apoptosis. Caspase-8, or FLICE, may be the first enzyme of the proteolytic cascade that is activated by the FAS ligand (FASL; 134638) and tumor necrosis factor (TNF; 191160). Caspase-8 is recruited to FAS (134637) and TNF receptor-1 (TNFR1; 191190) through interaction of its prodomain with the death effector domain (DED) of the receptor-associating protein FADD (602457). By searching EST databases for sequences related to FADD, Shu et al. (1997) identified cDNAs encoding a protein that they designated CASPER. The predicted 480-amino acid CASPER protein contains 2 DED-like modules at its N terminus and a C-terminal caspase-like protease domain. However, CASPER is not a caspase since it lacks several conserved amino acids found in all identified caspases. Northern blot analysis detected several CASPER transcripts, with highest expression in human skeletal muscle, pancreas, and heart.
The viral FLICE inhibitory proteins (v-FLIPs) contain 2 DEDs and block the early signaling events of the cellular death receptors. By searching EST databases for cellular homologs of v-FLIPs, Irmler et al. (1997) isolated human cDNAs encoding 2 isoforms of FLIP. FLIP(L), the longer isoform, contains 2 DEDs and a caspase-like domain, and FLIP(S), the shorter isoform, contains only the 2 DEDs followed by a C-terminal extension of approximately 50 amino acids.
Goltsev et al. (1997), Han et al. (1997), Hu et al. (1997), Inohara et al. (1997), and Srinivasula et al. (1997) isolated FLIP cDNAs; they designated the gene CASH (caspase homolog), MRIT (MACH-related inducer of toxicity; 'mrit' also means 'death' in Sanskrit), I-FLICE (inhibitor of FLICE), CLARP (caspase-like apoptosis regulatory protein), and FLAME1 (FADD-like antiapoptotic molecule-1), respectively. Han et al. (1997) cloned a cDNA encoding an MRIT isoform, which they called MRIT-alpha-2, that lacks the first DED.
Irmler et al. (1997) and Goltsev et al. (1997) isolated cDNAs encoding the mouse homolog of FLIP.
By microarray analysis, Jun et al. (2001) demonstrated expression of the CFLAR gene in human donor corneas.
Gene Function
Shu et al. (1997) showed that CASPER interacted with FADD, caspase-8 (601763), caspase-3 (CASP3; 600636), TRAF1 (601711), and TRAF2 (601895) through distinct domains. Overexpression of CASPER or its C-terminal protease-like domain potently induced apoptosis, whereas a deletion mutant lacking 45 C-terminal residues inhibited TNF- and FAS-induced apoptosis. Since this truncated form is encoded by a natural splice variant of CASPER, Shu et al. (1997) suggested that alternative splicing of CASPER may provide a mechanism to regulate apoptosis triggered by cell death pathways.
Irmler et al. (1997) found that activation of T cells induced a transient resistance to FAS-induced apoptotic signals that correlated with increased expression of FLIP(L). High levels of FLIP(L) protein were detected in melanoma cell lines and malignant melanoma tumors. The authors concluded that FLIP may be implicated in tissue homeostasis as an important regulator of apoptosis.
Han et al. (1997) found that, when expressed in mammalian cells, MRIT simultaneously and independently interacted with FLICE and BCLX(L) (600039), an antiapoptotic member of the BCL2 family. Han et al. (1997) suggested that MRIT may function as a link between cell survival and cell death pathways in mammalian cells.
In human islets, elevated glucose concentrations impair beta-cell proliferation and induce beta-cell apoptosis via upregulation of the FAS receptor. Maedler et al. (2002) observed expression of FLIP in human pancreatic beta-cells of nondiabetic individuals and decreased expression in tissue sections of type 2 diabetic patients. In vitro exposure of islets from nondiabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic beta-cells, in which FLIP was no longer detectable. Upregulation of FLIP, by incubation with transforming growth factor beta (TGFB1; 190180) or by transfection with an expression vector coding for FLIP, protected beta cells from glucose-induced apoptosis, restored beta-cell proliferation, and improved beta-cell function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-FAS antibody, indicating the dependence of these effects on FAS receptor activation. The data provided evidence for expression of FLIP in the human beta cell and suggested a novel approach to prevent and treat diabetes by switching FAS signaling from apoptosis to proliferation.
The biologic outcome of TNF treatment is determined by the balance between NF-kappa-B (see 164011), which promotes survival, and JNK (see 601158), which promotes cell death. Chang et al. (2006) found that Jnk activity controlled Tnf-induced cell death through proteasomal processing of Flip(L) in mice. Instead of direct phosphorylation of Flip(L), Jnk promoted accelerated decay of Flip(L) through phosphorylation and activation of the ubiquitin ligase Itch (606409). Jnk1 or Itch deficiency or treatment with a Jnk inhibitor rendered mice resistant in 3 distinct models of Tnf-induced liver failure, and cells from these mice did not show inducible Flip(L) ubiquitination and degradation. Chang et al. (2006) concluded that JNK antagonizes NF-kappa-B during TNF signaling by promoting proteasomal elimination of FLIP(L).
Oberst et al. (2011) showed that development of caspase-8-deficient mice is completely rescued by ablation of receptor-interacting protein kinase-3 (RIPK3; 605817). Adult animals lacking both caspase-8 and Ripk3 displayed a progressive lymphoaccumulative disease resembling that seen with defects in Cd95 (FAS; 134637) or Cd95 ligand (FASL; 134638), and resisted the lethal effects of Cd95 ligation in vivo. Oberst et al. (2011) found that caspase-8 prevents RIPK3-dependent necrosis without inducing apoptosis by functioning in a proteolytically active complex with CFLAR and that this complex is required for the protective function.
Lee et al. (2018) showed that cFLIP was necessary and sufficient for robust replication of hepatitis B virus (HBV; see 610424), as silencing of cFLIP downregulated HBV replication and expression of viral core and surface antigens in HepG2 hepatocytes. cFLIP interacted with and regulated the expression level of HBV X protein (HBx), which is required for HBV replication in hepatocytes, and cFLIP was essential for maintaining the steady-state level of HBx. cFLIP knockdown reduced the level of HBx by promoting its ubiquitin-dependent proteasomal degradation, thereby reducing HBV replication. Mutation analysis revealed that the DED1 domain of cFLIP was required to maintain HBx stability. Similar to HepG2 cells, cFLIP knockdown in Huh7 cells, in which HBx has no effect on HBV replication, also decreased HBV replication, indicating that cFLIP controls HBV replication through HBx -dependent and -independent pathways. Further analysis demonstrated that cFLIP regulated the expression or stability of hepatocyte nuclear factors (HNFs), which have critical roles in HBV transcription and maintenance of hepatocytes. In addition, knockdown of cFLIP in hepatocytes other than HepG2 cells also inhibited HBV replication, indicating that cFLIP is essential for viral replication during the natural course of HBV infection.
Muendlein et al. (2020) showed that deficiency of the long form (L) of cellular FLIP (cFLIP(L)) promotes mitochondrial complex II (see 600857) formation driving pyroptosis and the secretion of IL1-beta (147720) in response to lipopolysaccharide (LPS) alone. cFLIP(L) deficiency was sufficient to drive complex II formation in response to LPS. RIP1 (603453) and CASP8 (601763) recruitment to the FAS-associated death domain (FADD; 602457) occurred as early as 2 hours after LPS addition. Muendlein et al. (2020) found that in macrophages and perhaps in other cells, if levels of cFLIP(L) are sufficiently high, CASP8 activation and pyroptosis are inhibited. When cFLIP(L) levels are low, CASP8 homodimers form readily. Fully active CASP8 cleaves and activates distant targets, and LPS-activated macrophages rapidly undergo pyroptosis and secrete IL1-beta. CASP3 (600636), CASP7 (601761), and CASP9 (602234) are dispensable for CASP8-driven pyroptosis in the absence of cFLIP(L). Instead, CASP8 likely directly activates gasdermin D (GSDMD; 617042) to drive pyroptosis and the NLRP3 (606416) inflammasome to drive IL1-beta maturation and release.
By Western blot analysis, Mora-Molina et al. (2022) showed that cFLIP protein was downregulated in HCT116 colorectal cancer cells upon induction of endoplasmic reticulum (ER) stress. cFLIP downregulation was an early event in the signaling pathway, leading to caspase-8 activation in tumor cells undergoing ER stress. The cFLIP protein was downregulated via reduced activity of the protein synthesis machinery and proteasomal degradation of the remaining protein. Ectopic expression and knockdown analyses revealed that activation of caspase-8 and apoptosis upon ER stress depended on the levels of cFLIP(L), with a minor role for cFLIP(S). Moreover, the effects derived from cFLIP ectopic expression or knockdown resided downstream of activation of the PERK (EIF2AK3; 604032) branch of the unfolded protein response (UPR) pathway and of upregulation of TRAILR2 (TNFRSF10B; 603612), most likely by controlling the activation of caspase-8 at the intracellular death-inducing signaling complex (DISC) formed upon ER stress. Further analysis revealed that mTORC1 (601231) activity was substantially inhibited in HCT116 cells and that reduced mTORC1 activity likely protected cells from ER stress-induced cFLIP(L) loss and apoptosis.
By analyzing 293FT cells overexpressing mouse Usp27x (300975), Dold et al. (2022) showed that Usp27x sensitized 293FT cells to apoptosis through a contribution from an autocrine TNF loop that involved caspase-8 activation in response to apoptotic stimulation. Likewise, overexpression of mouse or human USP27X sensitized human melanoma cells to apoptosis mediated by caspase-8 in response to apoptotic stimulation. Analysis of apoptotic cell death in melanoma cells revealed that overexpression of human USP27X resulted in loss of cFLIP(L) protein through proteasomal degradation, and that the loss of cFLIP(L) protein promoted caspase-8 processing for its activation and apoptosis in response to apoptotic stimulation. cFLIP(L) was unlikely to be a direct target of USP27X deubiquitinating (DUB) activity, as cFLIPL formed a heterodimer with caspase-8 and heterodimerization was not affected by USP27XL overexpression. Moreover, USP27X interacted with components of the TNFR1/TLR3 (603029) DISC, including caspase-8, but not in combination with cFLIP. Further analysis suggested that USP27X enhanced substrate binding of the E3 ubiquitin ligase TRIM28 (601742) by releasing it from an inhibitory conformation through deubiquitination. Released TRIM28 was the required E3 ligase to target cFLIP(L) protein for degradation and apoptosis induction in response to stimulation. Other regulators of cFLIP expression were not involved in USP27X-dependent regulation of cFLIP and sensitization of cells to apoptosis.
Gene Structure
Hadano et al. (2001) determined that the CFLAR gene contains 14 exons and spans about 48 kb. It is transcribed in the centromere-to-telomere direction.
Mapping
Han et al. (1997) used fluorescence in situ hybridization and Srinivasula et al. (1997) used radiation hybrid mapping to localize the FLIP gene to 2q33-q34. Based on sequence similarity to STSs, Irmler et al. (1997) and Inohara et al. (1997) tentatively mapped the FLIP gene to 2q33. Irmler et al. (1997) noted that the FLIP gene colocalizes with caspase-10 (CASP10; 601762) on 2q33, suggesting that these genes arose by gene duplication. Hadano et al. (2001) determined that the CFLAR, CASP10, and CASP8 (601763) genes are tandemly located within 200 kb.
Animal Model
Yeh et al. (2000) observed that mice deficient in Cflar failed to survive beyond embryonic day 10.5 and exhibited impaired heart development, similar to mice lacking Fadd or Casp8. Unlike mice lacking Fadd or Casp8, however, Cflar -/- embryonic fibroblasts were highly sensitive to FASL- or TNF-induced apoptosis, showing rapid induction of Casp3 and Casp8 activities. Both nuclear factor kappa-B and Jnk/Sapk were activated in Cflar-deficient and wildtype cells in response to TNF. Yeh et al. (2000) proposed that CFLAR cooperates with CASP8 and FADD during embryonic development and regulates death factor-induced apoptosis induced by FAS or TNFR1 engagement.
Huang et al. (2010) generated mice with conditional loss of Flip expression in myeloid cells. These mice exhibited growth retardation, premature death, and splenomegaly with extramedullary hematopoiesis. They also showed increased circulating neutrophils with multiorgan neutrophil infiltration. Monocytes were also increased, but macrophages were reduced. In vitro, differentiation to macrophages was Flip dependent. Huang et al. (2010) concluded that FLIP is necessary for macrophage differentiation and homeostatic regulation of granulopoiesis.
REFERENCES
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Chang, L., Kamata, H., Solinas, G., Luo, J.-L., Maeda, S., Venuprasad, K., Liu, Y.-C., Karin, M. The E3 ubiquitin ligase Itch couples JNK activation to TNF-alpha-induced cell death by inducing c-FLIP(L) turnover. Cell 124: 601-613, 2006. [PubMed: 16469705] [Full Text: https://doi.org/10.1016/j.cell.2006.01.021]
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Dold, M. N., Ng, X., Alber, C., Gentle, I. E., Hacker, G., Weber, A. The deubiquitinase Usp27x as a novel regulator of cFLIPL protein expression and sensitizer to death-receptor-induced apoptosis. Apoptosis 27: 112-132, 2022. [PubMed: 35044632] [Full Text: https://doi.org/10.1007/s10495-021-01706-9]
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Goltsev, Y. V., Kovalenko, A. V., Arnold, E., Varfolomeev, E. E., Brodianskii, V. M., Wallach, D. CASH, a novel caspase homologue with death effector domains. J. Biol. Chem. 272: 19641-19644, 1997. [PubMed: 9289491] [Full Text: https://doi.org/10.1074/jbc.272.32.19641]
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Hadano, S., Yanagisawa, Y., Skaug, J., Fichter, K., Nasir, J., Martindale, D., Koop, B. F., Scherer, S. W., Nicholson, D. W., Rouleau, G. A., Ikeda, J.-E., Hayden, M. R. Cloning and characterization of three novel genes, ALS2CR1, ALS2CR2, and ALS2CR3, in the juvenile amyotrophic lateral sclerosis (ALS2) critical region at chromosome 2q33-q34: candidate genes for ALS2. Genomics 71: 200-213, 2001. [PubMed: 11161814] [Full Text: https://doi.org/10.1006/geno.2000.6392]
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Han, D. K. M., Chaudhary, P. M., Wright, M. E., Friedman, C., Trask, B. J., Riedel, R. T., Baskin, D. G., Schwartz, S. M., Hood, L. MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclX(L) and initiates cell death. Proc. Nat. Acad. Sci. 94: 11333-11338, 1997. [PubMed: 9326610] [Full Text: https://doi.org/10.1073/pnas.94.21.11333]
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Hu, S., Vincenz, C., Ni, J., Gentz, R., Dixit, V. M. I-FLICE, a novel inhibitor of tumor necrosis factor receptor-1- and CD-95-induced apoptosis. J. Biol. Chem. 272: 17255-17257, 1997. [PubMed: 9211860] [Full Text: https://doi.org/10.1074/jbc.272.28.17255]
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Huang, Q.-Q., Perlman, H., Huang, Z., Birkett, R., Kan, L., Agrawal, H., Misharin, A., Gurbuxani, S., Crispino, J. D., Pope, R. M. FLIP: a novel regulator of macrophage differentiation and granulocyte homeostasis. Blood 116: 4968-4977, 2010. [PubMed: 20724542] [Full Text: https://doi.org/10.1182/blood-2009-11-252841]
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Inohara, N., Koseki, T., Hu, Y., Chen, S., Nunez, G. CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis. Proc. Nat. Acad. Sci. 94: 10717-10722, 1997. [PubMed: 9380701] [Full Text: https://doi.org/10.1073/pnas.94.20.10717]
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Jun, A. S., Liu, S. H., Koo, E. H., Do, D. V., Stark, W. J., Gottsch, J. D. Microarray analysis of gene expression in human donor corneas. Arch. Ophthal. 119: 1629-1634, 2001. [PubMed: 11709013] [Full Text: https://doi.org/10.1001/archopht.119.11.1629]
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Lee, A. R., Lim, K. H., Park, E. S., Kim, D. H., Park, Y. K., Park, S., Kim, D. S., Shin, G. C., Kang, H. S., Won, J., Sim, H., Ha, Y. N., Jae, B., Choi, S. I., Kim, K. H. Multiple functions of cellular FLIP are essential for replication of hepatitis B virus. J. Virol. 92: e00339-18, 2018. [PubMed: 29875248] [Full Text: https://doi.org/10.1128/JVI.00339-18]
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Maedler, K., Fontana, A., Ris, F., Sergeev, P., Toso, C., Oberholzer, J., Lehmann, R., Bachmann, F., Tasinato, A., Spinas, G. A., Halban, P. A., Donath, M. Y. FLIP switches Fas-mediated glucose signaling in human pancreatic beta cells from apoptosis to cell replication. Proc. Nat. Acad. Sci. 99: 8236-8241, 2002. [PubMed: 12060768] [Full Text: https://doi.org/10.1073/pnas.122686299]
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Mora-Molina, R., Stohr, D., Rehm, M., Lopez-Rivas, A. cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells. Cell Death Dis. 13: 111, 2022. [PubMed: 35115486] [Full Text: https://doi.org/10.1038/s41419-022-04574-6]
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Muendlein, H. I., Jetton, D., Connolly, W. M., Eidell, K. P., Magri, Z., Smirnova, I., Poltorak, A. cFLIP(L) protects macrophages from LPS-induced pyroptosis via inhibition of complex II formation. Science 367: 1379-1384, 2020. [PubMed: 32193329] [Full Text: https://doi.org/10.1126/science.aay3878]
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Srinivasula, S. M., Ahmad, M., Ottilie, S., Bullrich, F., Banks, S., Wang, Y., Fernandes-Alnemri, T., Croce, C. M., Litwack, G., Tomaselli, K. J., Armstrong, R. C., Alnemri, E. S. FLAME-1, a novel FADD-like anti-apoptotic molecule that regulates Fas/TNFR1-induced apoptosis. J. Biol. Chem. 272: 18542-18545, 1997. [PubMed: 9228018] [Full Text: https://doi.org/10.1074/jbc.272.30.18542]
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Yeh, W.-C., Itie, A., Elia, A. J., Ng, M., Shu, H.-B., Wakeham, A., Mirtsos, C., Suzuki, N., Bonnard, M., Goeddel, D. V., Mak, T. W. Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development. Immunity 12: 633-642, 2000. [PubMed: 10894163] [Full Text: https://doi.org/10.1016/s1074-7613(00)80214-9]
Ada Hamosh - updated : 06/03/2020
Ada Hamosh - updated : 6/7/2011
Paul J. Converse - updated : 6/1/2011
Matthew B. Gross - updated : 5/21/2009
Jane Kelly - updated : 11/21/2002
Patricia A. Hartz - updated : 11/8/2002
Victor A. McKusick - updated : 7/3/2002
Paul J. Converse - updated : 4/23/2002
alopez : 06/03/2020
alopez : 06/14/2011
terry : 6/7/2011
mgross : 6/6/2011
terry : 6/1/2011
wwang : 5/28/2009
mgross : 5/21/2009
mgross : 5/21/2009
carol : 11/21/2002
mgross : 11/8/2002
cwells : 7/19/2002
cwells : 7/18/2002
terry : 7/3/2002
mgross : 4/23/2002
psherman : 5/7/1999
psherman : 3/2/1999