title: Balance between short and long isoforms of cFLIP regulates Fas-mediated apoptosis in vivo.
abstract: 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.
authors.affiliation: Program in Immunology, Sackler Graduate School, Tufts University, Boston, MA 02111;
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Ram
authors.affiliation: Institute of High-Tech Biomedicine, Petrozavodsk State University, Petrozavodsk, Republic of Karelia, 117198 Russia;
authors.forename: Vladimir
authors.initials: V
authors.lastname: Ilyukha
authors.affiliation: Institute of High-Tech Biomedicine, Petrozavodsk State University, Petrozavodsk, Republic of Karelia, 117198 Russia;
authors.forename: Tatyana
authors.initials: T
authors.lastname: Volkova
authors.affiliation: Laboratory of Bioinformatics, D. Rogachyov Federal Research Center for Pediatrics Hematology, Oncology and Immunology, Moscow, 117198 Russia; Centre for Convergence of Nano-, Bio-, Information and Cognitive Sciences and Technologies, National Research Centre "Kurchatov Institute," Moscow, 123182 Russia;
authors.forename: Anton
authors.initials: A
authors.lastname: Buzdin
authors.affiliation: Department of Integrative Physiology and Pathobiology, Tufts University School of Medicine, Boston, MA 02111.
authors.forename: Albert
authors.initials: A
authors.lastname: Tai
authors.affiliation: Department of Integrative Physiology and Pathobiology, Tufts University School of Medicine, Boston, MA 02111.
authors.forename: Irina
authors.initials: I
authors.lastname: Smirnova
authors.affiliation: Program in Immunology, Sackler Graduate School, Tufts University, Boston, MA 02111; Institute of High-Tech Biomedicine, Petrozavodsk State University, Petrozavodsk, Republic of Karelia, 117198 Russia; Department of Integrative Physiology and Pathobiology, Tufts University School of Medicine, Boston, MA 02111 Alexander.Poltorak@tufts.edu.
authors.forename: Alexander
authors.initials: A
authors.lastname: Poltorak
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D000906:Antibodies; D017209:Apoptosis; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics* / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D004198:Disease Susceptibility; D053222:Fas Ligand Protein / Q000378:metabolism; D008040:Genetic Linkage; D056426:Genetic Loci; D016678:Genome; D008099:Liver / Q000378:metabolism / Q000473:pathology; D008264:Macrophages / Q000378:metabolism; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D019655:Quantitative Trait, Heritable; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D000906:Antibodies; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D053222:Fas Ligand Protein; C491899:Fas protein, mouse; D020033:Protein Isoforms; D019014:fas Receptor; D053181:Caspase 8
keywords: Fas-receptor; apoptosis; cFLIP; caspase 8; liver failure