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Raf-1 sets the threshold of Fas sensitivity by modulating Rok-alpha signaling.

Piazzolla D, Meissl K, Kucerova L, Rubiolo C, Baccarini M - J. Cell Biol. (2005)

Bottom Line: Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-alpha.Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts.Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, Campus Vienna Biocenter, 1030 Vienna, Austria.

ABSTRACT
Ablation of the Raf-1 protein causes fetal liver apoptosis, embryonic lethality, and selective hypersensitivity to Fas-induced cell death. Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-alpha. In this study, we show that the kinase-independent modulation of Rok-alpha signaling is also the basis of the antiapoptotic function of Raf-1. Fas activation stimulates the formation of Raf-1-Rok-alpha complexes, and Rok-alpha signaling is up-regulated in Raf-1-deficient cells. This leads to increased clustering and membrane expression of Fas, which is rescued both by kinase-dead Raf-1 and by interfering with Rok-alpha or its substrate ezrin. Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts. Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.

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Heterozygosity at the lpr or gld locus prevents fetal liver apoptosis and embryonic lethality as a result of c-raf-1 ablation. (A) Heterozygosity at the lpr locus prevents fetal liver apoptosis in Raf-1 KO embryos. Parasagittal section of E11.5 livers from WT, Raf-1 KO, and Raf-1 KO;lpr/+ embryos. Note the presence of Fas clusters in Raf-1 KO fetal livers and the reduction in Fas staining in the Raf-1 KO;lpr/+ liver. Apoptotic (TUNEL+) cells are absent in Raf-1 KO;lpr/+. (B) Increased Fas surface expression in KO fetal liver cells. Fas expression (solid lines) was determined by FACS analysis as described in Fig. 1 B. Dashed lines, isotype controls. (C) Comparison of a Raf-1 KO;lpr/+ pup (right), a control littermate (left), and eyes open at birth phenotype of the Raf-1 KO;lpr/+ pup (right). The experiments were performed exclusively with F2 animals.
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fig7: Heterozygosity at the lpr or gld locus prevents fetal liver apoptosis and embryonic lethality as a result of c-raf-1 ablation. (A) Heterozygosity at the lpr locus prevents fetal liver apoptosis in Raf-1 KO embryos. Parasagittal section of E11.5 livers from WT, Raf-1 KO, and Raf-1 KO;lpr/+ embryos. Note the presence of Fas clusters in Raf-1 KO fetal livers and the reduction in Fas staining in the Raf-1 KO;lpr/+ liver. Apoptotic (TUNEL+) cells are absent in Raf-1 KO;lpr/+. (B) Increased Fas surface expression in KO fetal liver cells. Fas expression (solid lines) was determined by FACS analysis as described in Fig. 1 B. Dashed lines, isotype controls. (C) Comparison of a Raf-1 KO;lpr/+ pup (right), a control littermate (left), and eyes open at birth phenotype of the Raf-1 KO;lpr/+ pup (right). The experiments were performed exclusively with F2 animals.

Mentions: The major defect of the Raf-1 KO embryos is increased fetal liver apoptosis. Therefore, we investigated whether Fas expression was altered in this organ. At E11.5–12.5, the fetal liver consists of developing hepatocytes and haematopoetic cells, both of which reportedly express Fas (Terada and Nakanuma, 1995; De Maria et al., 1999). Consistent with the results obtained in PEFs and MEFs, Fas was often visualized as a rim around the cells in Raf-1–deficient livers (Fig. 7 A). In addition, FACS analysis showed that KO fetal liver cells expressed higher amounts of surface Fas compared with WT cells (Fig. 7 B). The lpr allele decreased Fas staining in both WT (not depicted) and KO fetal liver (Fig. 7, A and B). Raf-1 ablation is embryonic lethal both on the 129/Ola:Bl6 and on the 129/SvHsd:Bl6 background (Huser et al., 2001; Mikula et al., 2001). Remarkably, lpr heterozygosity rescued the fetal liver apoptosis that is characteristic of 129/SvHsd:Bl6 KO embryos (Fig. 7 A). The lpr allele did not significantly alter the ratio of E11.5 c-raf-1−/− embryos/litter, which was already submendelian (15%; n = 289) in this background (Huser et al., 2001; Mikula et al., 2001). This indicates the presence of an earlier defect with limited penetrance, and it could be connected with the placental insufficiency caused by Raf-1 ablation (Huser et al., 2001; Mikula et al., 2001). All c-raf-1−/−;lpr/+ embryos, however, survived to term, whereas their Raf-1–deficient littermates died within E12.5. Embryonic lethality could similarly be rescued by introducing a mutated fasl allele (Takahashi et al., 1994) in Raf-1 KO animals (unpublished data). Most likely as a consequence of the placental defects that persisted in the Raf-1 KO embryos heterozygous for lpr or gld (not depicted), the rescued Raf-1 KO pups were smaller than their control littermates, but they were not anemic (Fig. 7 C). Their eyes were consistently open at birth, which is a phenotype associated with delayed epithelial cell migration (Zhang et al., 2003) and consistent with the cell-autonomous migration defect of Raf-1 KO cells (Ehrenreiter et al., 2005). The rescued Raf-1 KO animals failed to thrive and died perinatally for reasons presently unknown. Thus, increased Fas surface expression correlates with liver apoptosis and embryonic lethality in Raf-1 KO embryos, and both can be relieved by reducing the amount of Fas. Like in fibroblasts, this does not rescue the defects associated with the cytoskeleton and migration, as the pups are born with their eyes open.


Raf-1 sets the threshold of Fas sensitivity by modulating Rok-alpha signaling.

Piazzolla D, Meissl K, Kucerova L, Rubiolo C, Baccarini M - J. Cell Biol. (2005)

Heterozygosity at the lpr or gld locus prevents fetal liver apoptosis and embryonic lethality as a result of c-raf-1 ablation. (A) Heterozygosity at the lpr locus prevents fetal liver apoptosis in Raf-1 KO embryos. Parasagittal section of E11.5 livers from WT, Raf-1 KO, and Raf-1 KO;lpr/+ embryos. Note the presence of Fas clusters in Raf-1 KO fetal livers and the reduction in Fas staining in the Raf-1 KO;lpr/+ liver. Apoptotic (TUNEL+) cells are absent in Raf-1 KO;lpr/+. (B) Increased Fas surface expression in KO fetal liver cells. Fas expression (solid lines) was determined by FACS analysis as described in Fig. 1 B. Dashed lines, isotype controls. (C) Comparison of a Raf-1 KO;lpr/+ pup (right), a control littermate (left), and eyes open at birth phenotype of the Raf-1 KO;lpr/+ pup (right). The experiments were performed exclusively with F2 animals.
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Related In: Results  -  Collection

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fig7: Heterozygosity at the lpr or gld locus prevents fetal liver apoptosis and embryonic lethality as a result of c-raf-1 ablation. (A) Heterozygosity at the lpr locus prevents fetal liver apoptosis in Raf-1 KO embryos. Parasagittal section of E11.5 livers from WT, Raf-1 KO, and Raf-1 KO;lpr/+ embryos. Note the presence of Fas clusters in Raf-1 KO fetal livers and the reduction in Fas staining in the Raf-1 KO;lpr/+ liver. Apoptotic (TUNEL+) cells are absent in Raf-1 KO;lpr/+. (B) Increased Fas surface expression in KO fetal liver cells. Fas expression (solid lines) was determined by FACS analysis as described in Fig. 1 B. Dashed lines, isotype controls. (C) Comparison of a Raf-1 KO;lpr/+ pup (right), a control littermate (left), and eyes open at birth phenotype of the Raf-1 KO;lpr/+ pup (right). The experiments were performed exclusively with F2 animals.
Mentions: The major defect of the Raf-1 KO embryos is increased fetal liver apoptosis. Therefore, we investigated whether Fas expression was altered in this organ. At E11.5–12.5, the fetal liver consists of developing hepatocytes and haematopoetic cells, both of which reportedly express Fas (Terada and Nakanuma, 1995; De Maria et al., 1999). Consistent with the results obtained in PEFs and MEFs, Fas was often visualized as a rim around the cells in Raf-1–deficient livers (Fig. 7 A). In addition, FACS analysis showed that KO fetal liver cells expressed higher amounts of surface Fas compared with WT cells (Fig. 7 B). The lpr allele decreased Fas staining in both WT (not depicted) and KO fetal liver (Fig. 7, A and B). Raf-1 ablation is embryonic lethal both on the 129/Ola:Bl6 and on the 129/SvHsd:Bl6 background (Huser et al., 2001; Mikula et al., 2001). Remarkably, lpr heterozygosity rescued the fetal liver apoptosis that is characteristic of 129/SvHsd:Bl6 KO embryos (Fig. 7 A). The lpr allele did not significantly alter the ratio of E11.5 c-raf-1−/− embryos/litter, which was already submendelian (15%; n = 289) in this background (Huser et al., 2001; Mikula et al., 2001). This indicates the presence of an earlier defect with limited penetrance, and it could be connected with the placental insufficiency caused by Raf-1 ablation (Huser et al., 2001; Mikula et al., 2001). All c-raf-1−/−;lpr/+ embryos, however, survived to term, whereas their Raf-1–deficient littermates died within E12.5. Embryonic lethality could similarly be rescued by introducing a mutated fasl allele (Takahashi et al., 1994) in Raf-1 KO animals (unpublished data). Most likely as a consequence of the placental defects that persisted in the Raf-1 KO embryos heterozygous for lpr or gld (not depicted), the rescued Raf-1 KO pups were smaller than their control littermates, but they were not anemic (Fig. 7 C). Their eyes were consistently open at birth, which is a phenotype associated with delayed epithelial cell migration (Zhang et al., 2003) and consistent with the cell-autonomous migration defect of Raf-1 KO cells (Ehrenreiter et al., 2005). The rescued Raf-1 KO animals failed to thrive and died perinatally for reasons presently unknown. Thus, increased Fas surface expression correlates with liver apoptosis and embryonic lethality in Raf-1 KO embryos, and both can be relieved by reducing the amount of Fas. Like in fibroblasts, this does not rescue the defects associated with the cytoskeleton and migration, as the pups are born with their eyes open.

Bottom Line: Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-alpha.Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts.Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.

View Article: PubMed Central - PubMed

Affiliation: Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, Campus Vienna Biocenter, 1030 Vienna, Austria.

ABSTRACT
Ablation of the Raf-1 protein causes fetal liver apoptosis, embryonic lethality, and selective hypersensitivity to Fas-induced cell death. Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-alpha. In this study, we show that the kinase-independent modulation of Rok-alpha signaling is also the basis of the antiapoptotic function of Raf-1. Fas activation stimulates the formation of Raf-1-Rok-alpha complexes, and Rok-alpha signaling is up-regulated in Raf-1-deficient cells. This leads to increased clustering and membrane expression of Fas, which is rescued both by kinase-dead Raf-1 and by interfering with Rok-alpha or its substrate ezrin. Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts. Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.

Show MeSH
Related in: MedlinePlus