Caspases are cysteine proteases that play a central role in apoptosis. Caspase-8 (FLICE; OMIM 601763) may be the first enzyme of the proteolytic cascade
that is activated by the FAS ligand (OMIM 134638) and tumor necrosis factor (TNF; OMIM 191160). By
searching EST databases for sequences related to FADD, CASPER
(caspase-eight-related protein) was found. 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.
NCBI Summary:
The protein encoded by this gene is a regulator of apoptosis and is structurally similar to caspase-8. However, the encoded protein lacks caspase activity and appears to be itself cleaved into two peptides by caspase-8. Several transcript variants encoding different isoforms have been found for this gene, and partial evidence for several more variants exists. [provided by RefSeq, Feb 2011]
General function
Cell death/survival, Anti-apoptotic, Apoptosis
Comment
Cellular localization
Cytoplasmic
Comment
Ovarian function
Follicle atresia, Oogenesis
Comment
cFLIP Regulates Death Receptor-mediated Apoptosis in an Ovarian Granulosa Cell Line by Inhibiting Procaspase-8 Cleavage. Matsuda F et al. More than 99% of follicles in mammalian ovaries undergo atresia, but the mechanisms regulating the strict selection process are still unclear. Granulosa cell apoptosis is considered the trigger of follicular atresia, which occurs in advance of the death of an oocyte. Cellular FLICE-like inhibitory protein (cFLIP), a homologue of procaspase-8 (also called FLICE), is an intracellular anti-apoptotic protein. It is expressed in granulosa cells of porcine ovaries, where its levels decreases during follicular atresia. We hypothesized that cFLIP regulates granulosa cell apoptosis by acting as a pro-survival factor. In the present study, to further reveal the function of cFLIP in granulosa cells, we examined the anti-apoptotic mechanism of cFLIP using KGN, a human granulosa tumor cell line. Fas-mediated apoptosis was induced by co-treatment with anti-Fas antibody (CH-11), which acts as an agonist of Fas-ligand, and cycloheximide (CHX). When cFLIP was stably expressed in KGN cells following transfection of an expression vector, the Fas-mediated apoptosis was inhibited. Suppression of cFLIP by small interfering RNA (siRNA) spontaneously induced cell death. Silencing of cFLIP promoted cleavage of procaspase-8, and the cell death caused by cFLIP siRNA was completely blocked by a caspase-8 inhibitor (Z-IETD-FMK), indicating that cFLIP regulates apoptosis in KGN cells by inhibiting cleavage of procaspase-8. In conclusion, cFLIP is an essential pro-survival factor for granulosa cells, and it prevents granulosa cell apoptosis by inhibiting procaspase-8 activation.
Changes in expression of anti-apoptotic protein, cflip, in granulosa cells during follicular atresia in porcine ovaries Matsuda-Minehata F, et al .
Follicular selection is performed in mammalian ovaries, as most follicles undergo atresia during follicular development and growth. Follicular regression is indicated to begin with granulosa cell apoptosis. To reveal the molecular mechanisms of the selection, we examined the changes in the levels of cellular-Flice like inhibitory protein (cFLIP) expression in porcine granulosa cells. cFLIP is the homologue of intracellular apoptosis inducer (procaspase-8/Flice), and has two alternative splicing isoforms: cFLIP short form (cFLIP(S)) and long form (cFLIP(L)). By competing with caspase-8, cFLIP inhibits apoptosis initiated by death receptors. The changes in the levels of cFLIP(S) and cFLIP(L) mRNA and protein expression in granulosa cells were determined by RT-PCR and Western blotting, respectively. cFLIP(L) mRNA and protein were highly expressed in granulosa cells of healthy follicles and decreased during atresia. cFLIP(S) mRNA levels in granulosa cells were low and showed no change among the stages of follicular development, and its protein level was extremely low. We examined the changes in the localization of cFLIP mRNAs in pig ovaries by in situ hybridization and found that cFLIP(L) is abundant in granulosa cells of healthy follicles in comparison with those of atretic follicles. Immunohistochemical analyses demonstrated that the cFLIP protein is highly expressed in the granulosa cell of healthy follicles but weakly expressed in that of atretic follicles. We presumed that cFLIP, especially cFLIP(L), plays an anti-apoptotic role in the granulosa cells of healthy follicles of pig ovaries, and that cFLIP could be a major survival factor that determines whether growth or atresia occurs in porcine follicles.
Anti-apoptotic activity of porcine cFLIP in ovarian granulosa cell lines. Matsuda-Minehata F et al. In mammalian ovaries, more than 99% of follicles undergo atresia during growth and development. Recently, we found that the expression of cellular FLICE-like inhibitory protein long form (cFLIP(L)) decreased during follicular atresia in granulosa cells of porcine ovaries. In humans and other species, both the short (cFLIP(S)) and long (cFLIP(L)) forms of cFLIP are considered to function as cell survival factors that inhibit death ligand receptor-mediated apoptosis. Since the anti-apoptotic activity of porcine cFLIP (pcFLIP) in granulosa cells had not been determined, we examined the effect of pcFLIP on survival using granulosa-derived cell lines. A human cervix adenocarcinoma cell line, HeLa, human ovarian granulosa tumor cell line, KGN, and porcine granulosa-derived cell line, JC-410, were used. By Western blotting, internal cFLIP(L) was detected in all cell lines, but only trace levels of cFLIP(S) were found in HeLa and KGN cells. To examine the anti-apoptotic activity, pcFLIP(S) or pcFLIP(L) was overexpressed in HeLa and KGN cells. Transfected cells in which pcFLIP(S) or pcFLIP(L) was overexpressed, survived the induction of Fas-mediated apoptosis, while almost all of the cells transfected with empty vector died. Then, we suppressed the expression of porcine cFLIP(S) and/or cFLIP(L) in JC-410 cells using small interfering RNA (siRNA). When both cFLIP(S) and cFLIP(L), or only cFLIP(L) was suppressed, cell viability declined significantly. From the results, we conclude that porcine cFLIP(S) and cFLIP(L) exhibit anti-apoptotic activity in granulosa-derived cells. It was strongly suggested that pcFLIP acts as a survival-promoting factor in granulosa cells and determines whether porcine ovarian follicles survive or undergo atresia. Mol. Reprod. Dev. 74: 1165-1170, 2007. (c) 2007 Wiley-Liss, Inc.
Expression regulated by
Growth Factors/ cytokines
Comment
Wu Xiao C, et al reported that nuclear factor kappaB-mediated induction of Flice-like inhibitory protein prevents tumor necrosis factor alpha-induced apoptosis in rat granulosa cells.
The purpose of the studies was to examine the role and regulation of the antiapoptotic Flice-like inhibitory protein (FLIP) in rat granulosa cells by tumor necrosis factor alpha (TNFalpha) in vitro. Granulosa cells from immature rats primed with eCG were cultured in serum-free RPMI in the absence or presence of TNFalpha (20 ng/ml), cycloheximide (CHX, 10 microg/ml), SN50 (a specific inhibitor of nuclear factor kappaB ,NFkappaB, translocation, 100 or 200 microg/ml), or a combination of these. (SM50, a mutated inactive peptide of SN50, was used as control.) Inhibitor kappaB (IkappaB; total and phosphorylated forms) and NFkappaB binding abilities were measured by Western blot and electrophoretic mobility shift assay, respectively. Apoptosis was assessed by in situ TUNEL assay, whereas FLIP mRNA levels were determined by semiquantitative reverse transcriptase-polymerase chain reaction. TNFalpha alone failed to induce granulosa cell death but significantly increased the apoptotic cell number in the presence of cycloheximide. TNFalpha significantly up-regulated the expression of the short form of FLIP (FLIP(S)) but not the long form (FLIP(L)). TNFalpha induced IkappaB phosphorylation and NFkappaB activation. SN50, but not SM50, attenuated TNFalpha-induced FLIP(S) expression and enhanced TNFalpha-induced apoptosis. Down-regulation of TNFalpha-induced FLIP(S) by FLIP(S) antisense expression enhanced TNFalpha-induced apoptosis. A full length of rat FLIP(S), with high homology to mouse FLIP(S) (85%), had been cloned and sequenced. These findings suggest that, in addition to its proapoptotic function, TNFalpha can induce an intracellular survival factor for the maintenance of follicular development. TNFalpha-induced, NFkappaB-mediated FLIP(S) expression is a determinant of granulosa cell fate.
The Role of Interleukin-6 in the Regulation of Granulosa Cell Apoptosis during Follicular Atresia in Pig Ovaries. Maeda A et al. More than 99% of follicles in mammalian ovaries undergo a degenerative process known as atresia, and only a few follicles actually ovulate during follicular growth and development. Follicular selection mostly depends on granulosa cell apoptosis, but the molecular mechanism behind the regulation of this selective atresia is still largely unknown. In the present study, to examine whether or not interleukin-6 (IL-6), a multifunctional cytokine, is involved in apoptosis during atresia in pig ovaries, the expression of IL-6 mRNA in granulosa cells was quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR). The level of mRNA decreased during atresia. Enzyme-linked immunosorbent assay (ELISA) showed that the level of IL-6 protein in follicular fluid also decreased during atresia. Moreover, recombinant human IL-6 upregulated the expression of an intracellular apoptosis inhibitor, cellular FLICE-like inhibitory protein long form (cFLIPL), in cultured cells derived from human granulosa cells. It is possible that IL-6 is produced in the granulosa cells of healthy follicles, that it increases the cFLIPL level, and cFLIPL then prevents apoptotic cell death.
Ovarian localization
Oocyte, Granulosa, Luteal cells
Comment
Stanton JL, et al 2002 reported the gene expression profiling of human GV oocytes based on an analysis of a
profile obtained by serial analysis of gene expression (SAGE).
A gene expression profile of the human GV oocyte has recently been established
by Serial Analysis of Gene Expression (SAGE). A significant number of the
genes identified in this profile had not previously been associated with
mammalian oocytes. We sought to confirm gene matches by RT-PCR amplification
of candidate transcripts using mouse eggs. Attention focused on receptors,
proteins involved in apoptosis, and cytoskeletal proteins. Two receptors found
in the human catalogue, CCR6 and PAR3, were not found in mouse eggs, whereas
myosin light chain, LLGL, beta -actin, 5HT receptor, bad, bak, DFF45, and
Caspase homologue (cash) were. Individual SAGEtags can match more than one
gene and, in some cases, more than ten. Examination of transcript sequences
that generate multiple gene assignments identified a common denominator of
short interspersed elements or Alu sequences. For reasons which are, as yet,
unclear, the human GV oocyte SAGE catalogue contains relatively high
abundances of SAGEtags in Alu sequences. This may reflect normal expression of
Alu-containing. genes in eggs or upregulated expression of Alu elements
following stress. The degeneracy of gene matches in SAGE generated by Alu
sequences makes independent confirmation of candidate genes essential.
Follicle stages
Antral, Corpus luteum
Comment
Changes in cFLIP expression in the corpus luteum throughout the estrous cycle of Shibagoats. Jin HZ et al. (2015) Changes in the expression of the anti-apoptotic protein cFLIP (cellular FLICE inhibitory protein) were examined in the caprine corpus luteum (CL), during development and subsequent maintenance. Corpora lutea at four different stages were collected from Shiba goats, to measure the expression of cFLIP mRNA, protein and immunolocalization. Expression of short form cFLIP (cFLIPS) mRNA was highest at the early CL stage, and decreased during late and regressed stages (P < 0.05). In contrast, long form cFLIP (cFLIPL) mRNA expression was high during early, mid and late stages, and only decreased at the regressed stage (P< 0.01). Protein expression of cFLIPS was highest at the late CL stage, and decreased at the regressed stage (P < 0.01). Protein expression of cFLIPL was highest at the early and mid CL stages, and decreased by the late and regressed stages (P < 0.01). Further expression of cFLIPL was higher at the early CL stage than at the mid stage (P < 0.01). cFLIP protein expression was detectable by immunostaining in the early, mid and late CL stages, but not at the regressed CL stage. These results are consistent with the hypothesis that cFLIP acts as a survival factor in the maintenance of CL function in goats.//////////////////
Expression and localization of cFLIP anti-apoptotic protein in the porcine corpus luteum and corpora albicans during the estrous cycle and pregnancy. Jin HZ et al. (2015) 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 preg-nancy. The CL and CA were collected at different stages of estrous to determine cFLIP immunolocalization, and mRNA and protein ex-pression. 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 sur-vival factor, and performs an anti-apoptotic function in the porcine CL.//////////////////
Expression and Localization of cFLIP, an Anti-apoptotic Factor, in the Bovine Corpus Luteum. Hojo T et al. The objective of the present study was to investigate the potential mechanisms regulating cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptotic factor, in the bovine corpus luteum (CL). Expression of cFLIP mRNA was highest at the developing stage and then decreased significantly during the mid, late and regressed stages (P<0.05). Western blot analysis revealed that expression of the long isoform of cFLIP (cFLIP(L)) protein was high during the early and developing luteal stages, remained steady during the mid and late luteal stages and then decreased significantly (P<0.05) by the regressed stage. However, the expression levels of the short isoform of cFLIP (cFLIPS) remained low during the early, developing and mid luteal stages. Immunostaining of cFLIP was strongest in the cytoplasm of luteal and non-luteal cells, including endothelial and immune cells, remained high during the early, developing and mid luteal stages and then decreased significantly (P<0.05) in the late and regressed luteal stages. Immunostaining of cFLIP was observed only in macrophage-like cells in the regressing CL. However, cultured mid luteal cells had a higher percentage of cFLIP-positive cells and a lower percentage of TUNEL-positive cells than luteal cells treated with tumor necrosis factor alpha (TNF)/interferon gamma (IFNG; P<0.01). These results indicate downregulation of cFLIP during structural luteal regression, suggesting that cFLIP plays a survival role in the bovine CL.
Porcine (Sus scrofa) Cellular FLICE-like Inhibitory Protein (cFLIP): Molecular Cloning and Comparison with the Human and Murine cFLIP.
Goto Y, et al .
To reveal the molecular regulation mechanism of selective follicular atresia in porcine ovaries, we isolated the porcine cDNA encoding cellular FLICE-like inhibitory protein (cFLIP), which inhibits death receptor-mediated apoptosis signal transduction. Two alternative splicing isoforms of cFLIP, porcine cellular FLIP-short form (pcFLIP(S), 642 bp and 214-aa) and -long form (pcFLIP(L), 1446 bp and 482-aa), were identified from a cDNA library prepared from follicular granulosa cells of pig ovaries. pcFLIP(S) and pcFLIP(L) indicated high identities with human and murine cFLIP, and both of them contain two tandem specific amino acid regions (death effector domain: DED) in their N-terminal, suggesting that pcFLIP(S) and pcFLIP(L) inhibit the death receptor-mediated apoptosis signal by binding to other pro-apoptotic factors mediated by DED. pcFLIP(S) contains a short C-terminal region, while pcFLIP(L) has a caspase-like domain in the C-terminal region. The reverse transcription-polymerase chain reaction analysis revealed that both pcFLIP(S) and pcFLIP(L) mRNAs were highly expressed in granulosa cells of healthy follicles, suggesting that these cFLIPs play important roles in the regulation mechanism of apoptosis in ovarian follicular granulosa cells. The present data will contribute to understanding of the physiological roles of cFLIPs in the apoptosis regulation in porcine tissues.