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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 locus rescues the apoptotic defects of Raf-1 KO PEFs in culture. (A) Fas expression is reduced in the lpr/+ background, but Fas is organized in clusters at the surface of Raf-1 KO PEFs. Cells in suspension were treated with αFas as described in Fig. 2 A. Fas clustering was detectable in 68 ± 3% of KO cells and in 31 ± 2% of c-raf-1–/–;lpr/+ cells. (B) Fas expression is reduced in the lpr/+ background. The expression of Raf-1, Fas, and FasL in whole PEF lysates was analyzed by immunoblotting. An ERK immunoblot is shown as a loading control. Molecular mass markers are shown in kilodaltons on the left. (C) FasL mRNA levels were determined by RT-PCR. The HPRT gene was used as a normalization control. −, negative control; M, DNA marker in base pairs. (D) c-raf-1–/–;lpr/+ (KO;lpr/+) PEFs successfully accumulate in culture. WT, WT;lpr/+, KO, and KO;lpr/+ PEFs were cultured in DME/10% FCS. Cell numbers were determined at the indicated times. The values are the mean ± SD (error bars) of four individual batches of PEFs/genotype, each assayed in triplicate. *, P < 0.04; **, P < 0.025; ***, P < 0.01 according to a t test, all compared with KO. (E) Spontaneous apoptosis in continuously growing WT, WT;lpr/+, KO, and KO;lpr/+ PEFs. Asynchronous cells were stained with propidium iodide, and their DNA content was determined by FACS analysis. The percentage of apoptotic cells (DNA content < 2n) is indicated. (F) PEFs were treated with 500 ng/ml αFas plus 1 μg/ml Chx for 22 h. Cell death was assessed as in Fig. 1 A. The values in E and F are the mean ± SD (error bars) of at least three individual batches of PEFs/genotype. (E) *, P < 0.01. (F) *, P < 0.025 according to a t test, all compared with KO. (G) Defective internalization in KO and KO;lpr/+ MEFs. Internalization was determined as described in Fig. 2 B and expressed as the percentage of the internalization occurring in WT cells. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.025 according to a t test, all compared with WT.
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fig6: Heterozygosity at the lpr locus rescues the apoptotic defects of Raf-1 KO PEFs in culture. (A) Fas expression is reduced in the lpr/+ background, but Fas is organized in clusters at the surface of Raf-1 KO PEFs. Cells in suspension were treated with αFas as described in Fig. 2 A. Fas clustering was detectable in 68 ± 3% of KO cells and in 31 ± 2% of c-raf-1–/–;lpr/+ cells. (B) Fas expression is reduced in the lpr/+ background. The expression of Raf-1, Fas, and FasL in whole PEF lysates was analyzed by immunoblotting. An ERK immunoblot is shown as a loading control. Molecular mass markers are shown in kilodaltons on the left. (C) FasL mRNA levels were determined by RT-PCR. The HPRT gene was used as a normalization control. −, negative control; M, DNA marker in base pairs. (D) c-raf-1–/–;lpr/+ (KO;lpr/+) PEFs successfully accumulate in culture. WT, WT;lpr/+, KO, and KO;lpr/+ PEFs were cultured in DME/10% FCS. Cell numbers were determined at the indicated times. The values are the mean ± SD (error bars) of four individual batches of PEFs/genotype, each assayed in triplicate. *, P < 0.04; **, P < 0.025; ***, P < 0.01 according to a t test, all compared with KO. (E) Spontaneous apoptosis in continuously growing WT, WT;lpr/+, KO, and KO;lpr/+ PEFs. Asynchronous cells were stained with propidium iodide, and their DNA content was determined by FACS analysis. The percentage of apoptotic cells (DNA content < 2n) is indicated. (F) PEFs were treated with 500 ng/ml αFas plus 1 μg/ml Chx for 22 h. Cell death was assessed as in Fig. 1 A. The values in E and F are the mean ± SD (error bars) of at least three individual batches of PEFs/genotype. (E) *, P < 0.01. (F) *, P < 0.025 according to a t test, all compared with KO. (G) Defective internalization in KO and KO;lpr/+ MEFs. Internalization was determined as described in Fig. 2 B and expressed as the percentage of the internalization occurring in WT cells. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.025 according to a t test, all compared with WT.

Mentions: If the amount of Fas they express is the basis of the hypersensitivity of KO cells to Fas-induced apoptosis, reducing Fas levels should rescue the defects of Raf-1–deficient PEFs in culture. To assay this, we prepared PEFs from 129/SvHsd:Bl6 Raf-1 KO embryos that were heterozygous for the lpr mutation, which functionally inactivates Fas (Watanabe-Fukunaga et al., 1992). In Raf-1 KO PEFs, Fas accumulated at the membrane and was often organized in small patches and clusters (Fig. 6 A and Fig. S2 B, available at http://www.jcb.org/cgi/content/full/jcb.200504137/DC1), as observed in immortalized fibroblasts (Figs. 2 A and 4 D). lpr heterozygosity reduced the amount of total and membrane-associated Fas (Fig. 6, A and B). Raf-1 KO PEFs are hypersensitive to Fas-induced cell death (Fig. 6 F and Fig. S2 A) and fail to accumulate in culture as a result of increased spontaneous apoptosis (Mikula et al., 2001). These defects were rescued in c-raf-1−/−;lpr/+ cells (Fig. 6, D–F; KO;lpr/+), indicating that they were caused by alterations in Fas signaling. Because continuously growing KO and WT PEFs constitutively produce indistinguishable amounts of FasL (Fig. 6, B and C), the increase in spontaneous cell death shown by KO PEFs can be ascribed to Fas hypersensitivity (Fig. 6 E). Although KO;lpr/+ PEFs were not hypersensitive to Fas stimulation, Fas internalization was still perturbed in these cells (Fig. 6 G), and some Fas clustering could still be observed (Fig. 6 A). This confirms that Fas hypersensitivity is secondary to the cytoskeletal defects and that the net amount of Fas present on the cell surface is the crucial factor determining the differences between KO and WT cells.


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 locus rescues the apoptotic defects of Raf-1 KO PEFs in culture. (A) Fas expression is reduced in the lpr/+ background, but Fas is organized in clusters at the surface of Raf-1 KO PEFs. Cells in suspension were treated with αFas as described in Fig. 2 A. Fas clustering was detectable in 68 ± 3% of KO cells and in 31 ± 2% of c-raf-1–/–;lpr/+ cells. (B) Fas expression is reduced in the lpr/+ background. The expression of Raf-1, Fas, and FasL in whole PEF lysates was analyzed by immunoblotting. An ERK immunoblot is shown as a loading control. Molecular mass markers are shown in kilodaltons on the left. (C) FasL mRNA levels were determined by RT-PCR. The HPRT gene was used as a normalization control. −, negative control; M, DNA marker in base pairs. (D) c-raf-1–/–;lpr/+ (KO;lpr/+) PEFs successfully accumulate in culture. WT, WT;lpr/+, KO, and KO;lpr/+ PEFs were cultured in DME/10% FCS. Cell numbers were determined at the indicated times. The values are the mean ± SD (error bars) of four individual batches of PEFs/genotype, each assayed in triplicate. *, P < 0.04; **, P < 0.025; ***, P < 0.01 according to a t test, all compared with KO. (E) Spontaneous apoptosis in continuously growing WT, WT;lpr/+, KO, and KO;lpr/+ PEFs. Asynchronous cells were stained with propidium iodide, and their DNA content was determined by FACS analysis. The percentage of apoptotic cells (DNA content < 2n) is indicated. (F) PEFs were treated with 500 ng/ml αFas plus 1 μg/ml Chx for 22 h. Cell death was assessed as in Fig. 1 A. The values in E and F are the mean ± SD (error bars) of at least three individual batches of PEFs/genotype. (E) *, P < 0.01. (F) *, P < 0.025 according to a t test, all compared with KO. (G) Defective internalization in KO and KO;lpr/+ MEFs. Internalization was determined as described in Fig. 2 B and expressed as the percentage of the internalization occurring in WT cells. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.025 according to a t test, all compared with WT.
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fig6: Heterozygosity at the lpr locus rescues the apoptotic defects of Raf-1 KO PEFs in culture. (A) Fas expression is reduced in the lpr/+ background, but Fas is organized in clusters at the surface of Raf-1 KO PEFs. Cells in suspension were treated with αFas as described in Fig. 2 A. Fas clustering was detectable in 68 ± 3% of KO cells and in 31 ± 2% of c-raf-1–/–;lpr/+ cells. (B) Fas expression is reduced in the lpr/+ background. The expression of Raf-1, Fas, and FasL in whole PEF lysates was analyzed by immunoblotting. An ERK immunoblot is shown as a loading control. Molecular mass markers are shown in kilodaltons on the left. (C) FasL mRNA levels were determined by RT-PCR. The HPRT gene was used as a normalization control. −, negative control; M, DNA marker in base pairs. (D) c-raf-1–/–;lpr/+ (KO;lpr/+) PEFs successfully accumulate in culture. WT, WT;lpr/+, KO, and KO;lpr/+ PEFs were cultured in DME/10% FCS. Cell numbers were determined at the indicated times. The values are the mean ± SD (error bars) of four individual batches of PEFs/genotype, each assayed in triplicate. *, P < 0.04; **, P < 0.025; ***, P < 0.01 according to a t test, all compared with KO. (E) Spontaneous apoptosis in continuously growing WT, WT;lpr/+, KO, and KO;lpr/+ PEFs. Asynchronous cells were stained with propidium iodide, and their DNA content was determined by FACS analysis. The percentage of apoptotic cells (DNA content < 2n) is indicated. (F) PEFs were treated with 500 ng/ml αFas plus 1 μg/ml Chx for 22 h. Cell death was assessed as in Fig. 1 A. The values in E and F are the mean ± SD (error bars) of at least three individual batches of PEFs/genotype. (E) *, P < 0.01. (F) *, P < 0.025 according to a t test, all compared with KO. (G) Defective internalization in KO and KO;lpr/+ MEFs. Internalization was determined as described in Fig. 2 B and expressed as the percentage of the internalization occurring in WT cells. The values represent the mean ± SD (error bars) of three independent experiments. *, P < 0.025 according to a t test, all compared with WT.
Mentions: If the amount of Fas they express is the basis of the hypersensitivity of KO cells to Fas-induced apoptosis, reducing Fas levels should rescue the defects of Raf-1–deficient PEFs in culture. To assay this, we prepared PEFs from 129/SvHsd:Bl6 Raf-1 KO embryos that were heterozygous for the lpr mutation, which functionally inactivates Fas (Watanabe-Fukunaga et al., 1992). In Raf-1 KO PEFs, Fas accumulated at the membrane and was often organized in small patches and clusters (Fig. 6 A and Fig. S2 B, available at http://www.jcb.org/cgi/content/full/jcb.200504137/DC1), as observed in immortalized fibroblasts (Figs. 2 A and 4 D). lpr heterozygosity reduced the amount of total and membrane-associated Fas (Fig. 6, A and B). Raf-1 KO PEFs are hypersensitive to Fas-induced cell death (Fig. 6 F and Fig. S2 A) and fail to accumulate in culture as a result of increased spontaneous apoptosis (Mikula et al., 2001). These defects were rescued in c-raf-1−/−;lpr/+ cells (Fig. 6, D–F; KO;lpr/+), indicating that they were caused by alterations in Fas signaling. Because continuously growing KO and WT PEFs constitutively produce indistinguishable amounts of FasL (Fig. 6, B and C), the increase in spontaneous cell death shown by KO PEFs can be ascribed to Fas hypersensitivity (Fig. 6 E). Although KO;lpr/+ PEFs were not hypersensitive to Fas stimulation, Fas internalization was still perturbed in these cells (Fig. 6 G), and some Fas clustering could still be observed (Fig. 6 A). This confirms that Fas hypersensitivity is secondary to the cytoskeletal defects and that the net amount of Fas present on the cell surface is the crucial factor determining the differences between KO and WT cells.

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