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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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Selective hypersensitivity of Raf-1–deficient MEFs to Fas activation correlates with increased Fas expression. (A) Raf-1 KO MEFs are hypersensitive toward apoptosis induced by an agonistic Fas antibody or by FasL, but not by TNFα. MEFs were treated either with αFas, with recombinant FLAG-tagged FasL cross-linked with 1 μg/ml α-FLAG M2 antibody, or with recombinant mouse TNFα at the concentrations indicated for 22 h in the presence of 5 μg/ml Chx and 0.5% FCS. Cell death was determined by CytoTox 96 assay. The values represent the mean ± SD (error bars) of three independent cell lines. *, P < 0.02; **, P < 0.01, according to a t test comparing KO with WT cells. (B) The surface expression of Fas but not of TNFRI is altered in Raf-1 KO MEFs. WT and KO cells were stained with FITC-conjugated αFas (left) or with hamster α-mouse TNFRI antibody followed by FITC-conjugated goat α-hamster antibody (right) and were analyzed by flow cytometry. Dashed lines, isotype control (iso). (C and D) Fas is slightly overexpressed in Raf-1–deficient cells. (C) Different amounts of whole cell lysates from WT and KO cells were analyzed by αFas immunoblotting. Ponceau staining of the membrane is shown as a loading control. (D) Fas mRNA levels were determined by RT-PCR. The HPRT gene was used as a normalization control. −, negative control; M, DNA marker. Molecular mass markers (in kilodaltons, C; or bp, D) are shown on the left.
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fig1: Selective hypersensitivity of Raf-1–deficient MEFs to Fas activation correlates with increased Fas expression. (A) Raf-1 KO MEFs are hypersensitive toward apoptosis induced by an agonistic Fas antibody or by FasL, but not by TNFα. MEFs were treated either with αFas, with recombinant FLAG-tagged FasL cross-linked with 1 μg/ml α-FLAG M2 antibody, or with recombinant mouse TNFα at the concentrations indicated for 22 h in the presence of 5 μg/ml Chx and 0.5% FCS. Cell death was determined by CytoTox 96 assay. The values represent the mean ± SD (error bars) of three independent cell lines. *, P < 0.02; **, P < 0.01, according to a t test comparing KO with WT cells. (B) The surface expression of Fas but not of TNFRI is altered in Raf-1 KO MEFs. WT and KO cells were stained with FITC-conjugated αFas (left) or with hamster α-mouse TNFRI antibody followed by FITC-conjugated goat α-hamster antibody (right) and were analyzed by flow cytometry. Dashed lines, isotype control (iso). (C and D) Fas is slightly overexpressed in Raf-1–deficient cells. (C) Different amounts of whole cell lysates from WT and KO cells were analyzed by αFas immunoblotting. Ponceau staining of the membrane is shown as a loading control. (D) Fas mRNA levels were determined by RT-PCR. The HPRT gene was used as a normalization control. −, negative control; M, DNA marker. Molecular mass markers (in kilodaltons, C; or bp, D) are shown on the left.

Mentions: Raf-1–deficient mouse embryonic fibroblasts (MEFs) are selectively hypersensitive to apoptosis induced by Fas activation and showed altered Fas surface expression. 129/SvHsd:Bl6 Raf-1 KO MEFs are more sensitive than wild-type (WT) cells to apoptosis induced by an agonist αFas antibody (Mikula et al., 2001) as well as to the physiological stimulus Fas ligand (FasL; Fig. 1 A, top and middle). In both cases, Fas hypersensitivity could be detected over a broad range of doses. In contrast, Raf-1 KO and WT cells responded in an indistinguishable manner to TNFα over a broad range of concentrations (Fig. 1 A, bottom).


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)

Selective hypersensitivity of Raf-1–deficient MEFs to Fas activation correlates with increased Fas expression. (A) Raf-1 KO MEFs are hypersensitive toward apoptosis induced by an agonistic Fas antibody or by FasL, but not by TNFα. MEFs were treated either with αFas, with recombinant FLAG-tagged FasL cross-linked with 1 μg/ml α-FLAG M2 antibody, or with recombinant mouse TNFα at the concentrations indicated for 22 h in the presence of 5 μg/ml Chx and 0.5% FCS. Cell death was determined by CytoTox 96 assay. The values represent the mean ± SD (error bars) of three independent cell lines. *, P < 0.02; **, P < 0.01, according to a t test comparing KO with WT cells. (B) The surface expression of Fas but not of TNFRI is altered in Raf-1 KO MEFs. WT and KO cells were stained with FITC-conjugated αFas (left) or with hamster α-mouse TNFRI antibody followed by FITC-conjugated goat α-hamster antibody (right) and were analyzed by flow cytometry. Dashed lines, isotype control (iso). (C and D) Fas is slightly overexpressed in Raf-1–deficient cells. (C) Different amounts of whole cell lysates from WT and KO cells were analyzed by αFas immunoblotting. Ponceau staining of the membrane is shown as a loading control. (D) Fas mRNA levels were determined by RT-PCR. The HPRT gene was used as a normalization control. −, negative control; M, DNA marker. Molecular mass markers (in kilodaltons, C; or bp, D) are shown on the left.
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fig1: Selective hypersensitivity of Raf-1–deficient MEFs to Fas activation correlates with increased Fas expression. (A) Raf-1 KO MEFs are hypersensitive toward apoptosis induced by an agonistic Fas antibody or by FasL, but not by TNFα. MEFs were treated either with αFas, with recombinant FLAG-tagged FasL cross-linked with 1 μg/ml α-FLAG M2 antibody, or with recombinant mouse TNFα at the concentrations indicated for 22 h in the presence of 5 μg/ml Chx and 0.5% FCS. Cell death was determined by CytoTox 96 assay. The values represent the mean ± SD (error bars) of three independent cell lines. *, P < 0.02; **, P < 0.01, according to a t test comparing KO with WT cells. (B) The surface expression of Fas but not of TNFRI is altered in Raf-1 KO MEFs. WT and KO cells were stained with FITC-conjugated αFas (left) or with hamster α-mouse TNFRI antibody followed by FITC-conjugated goat α-hamster antibody (right) and were analyzed by flow cytometry. Dashed lines, isotype control (iso). (C and D) Fas is slightly overexpressed in Raf-1–deficient cells. (C) Different amounts of whole cell lysates from WT and KO cells were analyzed by αFas immunoblotting. Ponceau staining of the membrane is shown as a loading control. (D) Fas mRNA levels were determined by RT-PCR. The HPRT gene was used as a normalization control. −, negative control; M, DNA marker. Molecular mass markers (in kilodaltons, C; or bp, D) are shown on the left.
Mentions: Raf-1–deficient mouse embryonic fibroblasts (MEFs) are selectively hypersensitive to apoptosis induced by Fas activation and showed altered Fas surface expression. 129/SvHsd:Bl6 Raf-1 KO MEFs are more sensitive than wild-type (WT) cells to apoptosis induced by an agonist αFas antibody (Mikula et al., 2001) as well as to the physiological stimulus Fas ligand (FasL; Fig. 1 A, top and middle). In both cases, Fas hypersensitivity could be detected over a broad range of doses. In contrast, Raf-1 KO and WT cells responded in an indistinguishable manner to TNFα over a broad range of concentrations (Fig. 1 A, bottom).

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