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Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene

2001; Springer Nature; Volume: 20; Issue: 8 Linguagem: Inglês

10.1093/emboj/20.8.1952

1460-2075

Mario Mikula, Martin Schreiber, Zvenyslava Husak, Lucia Kučerová, J. Rüth, Rotraud Wieser, Kurt Zatloukal, Hartmut Beug, Erwin F. Wagner, Manuela Baccarini,

NF-κB Signaling Pathways

Article17 April 2001free access Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene Mario Mikula Mario Mikula Department of Cell- and Microbiology, Institute of Microbiology and Genetics, A-8036 Graz, Austria Search for more papers by this author Martin Schreiber Martin Schreiber Present address: Department of Obstetrics and Gynecology, University of Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria Search for more papers by this author Zvenislava Husak Zvenislava Husak Department of Cell- and Microbiology, Institute of Microbiology and Genetics, A-8036 Graz, Austria Search for more papers by this author Lucia Kucerova Lucia Kucerova Department of Cell- and Microbiology, Institute of Microbiology and Genetics, A-8036 Graz, Austria Search for more papers by this author Jochen Rüth Jochen Rüth Department of Cell- and Microbiology, Institute of Microbiology and Genetics, A-8036 Graz, Austria Search for more papers by this author Rotraud Wieser Rotraud Wieser Present address: Department of Medical Biology, University of Vienna, Währinger Straße 10, A-1090 Vienna, Austria Search for more papers by this author Kurt Zatloukal Kurt Zatloukal Department of Pathology, University of Graz, A-8036 Graz, Austria Search for more papers by this author Hartmut Beug Hartmut Beug Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria Search for more papers by this author Erwin F. Wagner Erwin F. Wagner Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria Search for more papers by this author Manuela Baccarini Corresponding Author Manuela Baccarini Department of Cell- and Microbiology, Institute of Microbiology and Genetics, A-8036 Graz, Austria Search for more papers by this author Mario Mikula Mario Mikula Department of Cell- and Microbiology, Institute of Microbiology and Genetics, A-8036 Graz, Austria Search for more papers by this author Martin Schreiber Martin Schreiber Present address: Department of Obstetrics and Gynecology, University of Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria Search for more papers by this author Zvenislava Husak Zvenislava Husak Department of Cell- and Microbiology, Institute of Microbiology and Genetics, A-8036 Graz, Austria Search for more papers by this author Lucia Kucerova Lucia Kucerova Department of Cell- and Microbiology, Institute of Microbiology and Genetics, A-8036 Graz, Austria Search for more papers by this author Jochen Rüth Jochen Rüth Department of Cell- and Microbiology, Institute of Microbiology and Genetics, A-8036 Graz, Austria Search for more papers by this author Rotraud Wieser Rotraud Wieser Present address: Department of Medical Biology, University of Vienna, Währinger Straße 10, A-1090 Vienna, Austria Search for more papers by this author Kurt Zatloukal Kurt Zatloukal Department of Pathology, University of Graz, A-8036 Graz, Austria Search for more papers by this author Hartmut Beug Hartmut Beug Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria Search for more papers by this author Erwin F. Wagner Erwin F. Wagner Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria Search for more papers by this author Manuela Baccarini Corresponding Author Manuela Baccarini Department of Cell- and Microbiology, Institute of Microbiology and Genetics, A-8036 Graz, Austria Search for more papers by this author Author Information Mario Mikula1, Martin Schreiber2, Zvenislava Husak1, Lucia Kucerova1, Jochen Rüth1, Rotraud Wieser3, Kurt Zatloukal4, Hartmut Beug5, Erwin F. Wagner5 and Manuela Baccarini 1 1Department of Cell- and Microbiology, Institute of Microbiology and Genetics, A-8036 Graz, Austria 2Present address: Department of Obstetrics and Gynecology, University of Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria 3Present address: Department of Medical Biology, University of Vienna, Währinger Straße 10, A-1090 Vienna, Austria 4Department of Pathology, University of Graz, A-8036 Graz, Austria 5Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1952-1962https://doi.org/10.1093/emboj/20.8.1952 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Raf kinases play a key role in relaying signals elicited by mitogens or oncogenes. Here, we report that c-raf-1−/− embryos are growth retarded and die at midgestation with anomalies in the placenta and in the fetal liver. Although hepatoblast proliferation does not appear to be impaired, c-raf-1−/− fetal livers are hypocellular and contain numerous apoptotic cells. Similarly, the poor proliferation of Raf-1−/− fibroblasts and hematopoietic cells cultivated in vitro is due to an increase in the apoptotic index of these cultures rather than to a cell cycle defect. Furthermore, Raf-1- deficient fibroblasts are more sensitive than wild- type cells to specific apoptotic stimuli, such as actinomycin D or Fas activation, but not to tumor necrosis factor-α. MEK/ERK activation is normal in Raf-1-deficient cells and embryos, and is probably mediated by B-Raf. These results indicate that the essential function of Raf-1 is to counteract apoptosis rather than to promote proliferation, and that effectors distinct from the MEK/ERK cascade must mediate the anti-apoptotic function of Raf-1. Introduction Cytosolic serine/threonine kinases convert extracellular stimuli into specific regulatory events affecting the pattern of gene expression, probably via phosphorylation of specific transcription factors. These kinases are often organized in cascades, a set-up that ensures signal modulation and amplification. The basic arrangement includes a small G-protein working upstream of a core module consisting of three kinases: a mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) that phosphorylates and activates a MAP kinase kinase (MAPKK), which in turn activates MAP kinase (MAPK). Activated Raf is the MAPKKK that regulates the ERK pathway, by phosphorylating and activating MEK. Within the MAPK cascade, Raf interacts physically with MEK-1 via its kinase domain and with GTP-loaded Ras via its N-terminus (Gu et al., 1994). Activated Ras binds to Raf with high affinity and mediates its translocation from the cytosol to the plasma membrane, where activation occurs via a complex, yet incompletely defined mechanism involving phosphorylation (Marshall, 1995; McCormick, 1995). Both Ras and Raf are proto-oncogenes. Thus, Raf represents an important intermediate in the transduction of regulated and deregulated proliferative signals (Naumann et al., 1997), and an attractive target for novel therapies aimed at interfering with its activation process and at eventually reversing its deregulated functions. Raf is a family of three serine/threonine-specific kinases (A-Raf, B-Raf and Raf-1) ubiquitously expressed throughout embryonic development. The three Raf isoforms are regulated differentially by upstream activators and exhibit quantitative differences in their ability to activate MEK (Marais et al., 1997), their best studied downstream substrate (Morrison and Cutler, 1997; Schaeffer and Weber, 1999). A-raf- and B-raf-deficient mice have been generated. Newborn A-raf−/− mice and B-raf−/− embryos are growth retarded, confirming the positive role of Raf kinases in cell proliferation suggested by cell culture experiments. However, while B-raf-deficient embryos die at midgestation because of vascular defects due to apoptotic death of differentiated endothelial cells (Wojnowski et al., 1997), A-raf-deficient mice show neurological and intestinal defects, depending on the genetic background (Pritchard et al., 1996). These divergent phenotypes show that Raf isoforms can not always compensate for each other and that they serve distinct functions in different tissues. The ubiquitously expressed c-raf-1 is certainly the best studied, but probably least understood Raf isoform. Mutation of c-raf-1 in the mouse, yielding an aberrant 62 kDa protein with residual kinase activity, results in embryonic or perinatal lethality, depending on the genetic background. The mutant embryos are growth retarded and display a rather complex phenotype with defects in the placenta, skin and lungs (Wojnowski et al., 1998). Here, we report the generation of a null mutation in the c-raf-1 gene in the mouse, which yields a recessive lethal phenotype. The embryos are growth retarded and die progressively around midgestation (E11.5–E13.5) with defects in the placenta and in the liver. The fetal liver is hypocellular and contains numerous apoptotic cells. In vitro studies with fibroblasts and hematopoietic cells confirmed that the non-redundant function of Raf-1 is to inhibit apoptosis, rather than to promote proliferation. ERK activation is not affected in Raf-1-deficient cells and embryos, indicating that the phenotypes observed are due to lack of activation of Raf-1-specific effectors distinct from the ERK pathway. Results Disruption of the c-raf-1 gene To inactivate the c-raf-1 gene, we constructed a vector containing loxP sites 5′ and 3′ of exon 3. A third loxP site as well as selection markers (a neomycin resistance gene for positive selection and the HSV thymidine kinase gene for negative selection) were inserted upstream of the floxed exon 3 (Figure 1A). The mutation was introduced into E14.1 embryonic stem (ES) cells by homologous recombination, and exon 3 and the Neo/TK gene cassette were then deleted by transiently expressing Cre. Positive clones were identified by Southern blot analysis (Figure 1B). Germline-transmitting chimeras were obtained and bred to 129/Sv and C57BL/6 mice. Offspring and implants were genotyped by PCR analysis (Figure 1C). The excision of exon 3 resulted in the complete loss of Raf-1, as shown by western blot analysis of primary c-raf-1−/− fibroblasts (Figure 1D). This result was confirmed using antibodies directed against the N- and C-terminal regions of Raf-1 (data not shown). Consistently, these cells were devoid of Raf-1 kinase activity, as determined by immune complex kinase assays (Figure 1E). Figure 1.Targeting of the mouse c-raf-1 gene. (A) Schematic representation of the targeting strategy. Genomic, genomic locus before recombination; targeted, homologously recombined targeting vector; null, targeted locus after Cre-mediated removal of exon 3 and of the Neo/TK cassette. The targeting vector contained loxP sites (triangles) 5′ and 3′ of exon 3 of the c-raf-1 gene as well as selection markers positioned between two loxP sites upstream of the floxed exon 3. The PstI sites delimiting the diagnostic fragments obtained by digesting the genomic and mutated c-raf-1 alleles are marked with an asterisk. Open arrows indicate the positions of the PCR primers 1, 2 and 3 used for genotyping. (B) Southern blot analysis of PstI-digested genomic DNA from a targeted ES cell clone. The probe used is indicated in (A). (C) PCR analysis of DNA isolated from E12.5 fetuses using the indicated primers. (D) Western blot of whole-cell lysates from MEFs using an antibody that recognizes the Raf kinase domain. (E) Immune complex kinase assay of Raf-1 immunoprecipitates from control (+/+) and Raf-1-deficient (−/−) fibroblasts untreated or stimulated with EGF (5 min, 33 nM). One representative experiment out of three is shown. Download figure Download PowerPoint Phenotype of c-raf-1−/− embryos No viable c-raf-1−/− offspring were born from heterozygous (c-raf-1+/−) intercrosses; therefore, c-raf-1 is essential for mouse development. To assess the time of death, we collected and genotyped conceptuses at earlier times in development. This analysis was carried out on an inbred 129/Sv background as well as on a mixed 129BL/6 background. The viability plot (Figure 2A) shows a progressive decrease in the percentage of c-raf-1−/− embryos from day 11.5 (E11.5) until E16.5. The window of lethality was broader on a mixed background, but none of the mutants survived past E16.5. c-raf-1−/− fetuses could be distinguished from their littermates starting from E11.5. The mutant fetuses of both inbred 129/Sv and mixed 129BL/6 background were smaller (∼20% weight reduction by E12.5; Figure 2B and data not shown) than wild-type or heterozygous littermates, and were developmentally retarded. Mutant placentas of both inbred 129/Sv and mixed 129BL/6 background were also smaller than those of wild-type or heterozygous littermates (∼20% weight reduction by E12.5). Histological examination revealed that both the spongiotrophoblast and the labyrinth layer of the c-raf-1−/− placentas were reduced in size. In particular, the labyrinth layer was poorly vascularized and contained abundant mesenchymal cells (Figure 2B and C and data not shown). These anomalies may affect the exchange of gases and nutrients between the fetus and the mother, thereby contributing to midgestational death. Figure 2.c-raf-1 inactivation is lethal at midgestation. (A) The percentage recovery of c-raf-1−/− conceptuses (of total viable embryos recovered from c-raf-1+/− intercrosses) is shown as a function of gestational age. Two hundred embryos of either background were genotyped. (B) Phenotype of a c-raf-1−/− fetus (right) and a control littermate (left) of 129/sv background at E12.5. c-raf-1−/− fetuses are smaller, developmentally retarded and paler than their normal littermates. The fetuses are shown with the placenta attached. (C) Placental defects in c-raf-1−/− mice. Radial sections of an E12.5 placenta from a wild-type (+/+) and a c-raf-1−/− littermate (−/−) of 129/sv background are shown. In the −/− placenta, the labyrinth layer (La) and the spongiotrophoblast layer (Sp) are reduced in size compared with +/+ controls. In addition, the labyrinth layer of the −/− placenta is less vascularized. In contrast, the decidua (De) appears normal. The lower panels show a higher magnification of the boxed areas. Note the mesenchymal cells that predominate in the −/− labyrinth layer (arrows). Download figure Download PowerPoint Increased apoptosis in c-raf-1−/− fetal livers In addition to their small size, a characteristic feature of the c-raf-1−/− fetuses was that their vasculature was less obvious and that they were paler than their littermates (Figure 2B). In particular, the mutant livers were very pale and significantly reduced in size. Histological examination revealed that the mutant liver was hypocellular compared with those of normal littermates, and that the sinuses contained fewer red blood cells (Figure 3A). In addition, cell size was slightly larger in the mutant embryos (Figure 3). This phenotype was observed in seven out of seven fetuses analyzed, of both inbred 129/Sv and mixed 129BL/6 background. Five of these seven mutant fetal livers contained large numbers of pyknotic and fragmented nuclei (Figure 3A). This morphology is typically associated with apoptosis. In situ end-labeling of DNA (TUNEL) confirmed elevated apoptosis in mutant livers (Figure 3B). The cell type affected was characterized by immunohistochemical staining using antibodies against keratins 8 and 18 (expressed in hepatoblasts) and against TER119 (erythroid specific). Pyknotic nuclei were found mainly in keratin 8- and keratin 18-positive and TER119-negative cells (Figure 3C and D). Thus, apoptosis in c-raf-1−/− fetal livers appears to be associated mainly with the hepatoblast compartment. Figure 3.Fetal liver defects in E12.5 c-raf-1−/− embryos. (A) Parasagittal section through the fetal liver of a c-raf-1−/− embryo (−/−) and of a littermate control (+/+) of 129/sv background. Note the hypocellularity and the abundance of pyknotic nuclei in the −/− livers. The boxed areas show higher magnifications of the fetal livers. (B) TUNEL analysis on sections from the same fetal livers shown in (A). Note the significant numbers of positively stained cells in the −/− liver. Sections were stained with propidium iodide to visualize the nuclei. (C and D) Immunohistochemical analysis of the fetal liver. The cell types undergoing apoptosis were characterized using rabbit polyclonal antibodies to keratins 8 and 18 to visualize hepatoblasts (C) and the erythroid-specific TER119 antibody (D). Note the presence of pyknotic and fragmented nuclei (white arrows) in keratin 8/18-positive and in TER119-negative cells of the −/− fetal liver. (E) In situ staining for the expression of PCNA; 81% of the wild-type and 88% of the −/− liver cells express PCNA, indicating that they are actively cycling. Download figure Download PowerPoint To ascertain whether a proliferation defect contributed to liver hypocellularity, we performed in situ staining for expression of proliferating cell nuclear antigen (PCNA). Most of the hepatoblasts had entered the cell cycle and expressed PCNA in both c-raf-1−/− embryos and normal littermates (Figure 3E). Analysis of c-raf-1−/− hematopoietic cells The above data imply that the anemic appearance of the fetuses might be due to the failure of the hepatoblasts to support hematopoiesis. It should be noted, however, that cells in the advanced stages of apoptosis may lose surface markers, and therefore negative results obtained by immunohistochemical staining are not conclusive. To assess directly whether the hematopoietic cells had a cell-autonomous survival defect, we established cultures of multipotent hematopoietic precursors from c-raf-1−/− and wild-type fetal livers in a serum-free medium (StemPro34), supplemented with stem cell factor (SCF), flk2/flt 3 ligand, interleukin (IL)-3, IL-6, granulocyte–macrophage colony-stimulating factor (GM-CSF) and dexamethasone. c-raf-1−/− fetal livers yielded low amounts of cells (1.96 × 106 compared with 14.7 × 106 obtained from +/+ littermates). These cells could be maintained in culture for up to 10 days. At this time, a total of 4530 × 106 cells had been generated from a single +/+ fetal liver, yielding a 308-fold net increase in cell number. Heterozygous livers yielded similar results. In contrast, only 121 × 106 multipotent cells could be recovered from the c-raf-1−/− fetal liver (61.7-fold net increase; Figure 4A). The experiment was repeated twice using single fetal livers or pools of three, with similar results. In Raf-1−/− cultures, the number of S-phase cells was only slightly reduced compared with wild type, but the number of apoptotic cells was increased significantly (Figure 4B; 17.46% as compared with 3.14% in wild-type cultures). Thus, the increase in spontaneous apoptosis most probably accounts for the defect observed. Figure 4.Analysis of c-raf-1−/− hematopoietic cells in culture. Fetal livers were isolated on E11.5 from wild-type (+/+) and homozygous c-raf-1−/− (−/−) littermates of mixed 129BL/6 background. Cells were pre-cultured in media supporting the expansion of hematopoietic cells for 8 days to enrich for multipotent hematopoietic precursors. (A) Multipotent precursors of each genotype were seeded at a density of 2 × 106 cells/ml, further cultivated and counted at daily intervals. Cells were maintained at a constant density of 2 × 106 cells/ml. The plot shows the total number of cells generated per fetal liver as a function of the time in culture. (B) Percentage of annexin V-positive (left panel) or BrdU-positive (right panel) cells in asynchronous cultures (day 8) of hematopoietic fetal livers cells from wild-type (+/+, open bars) and c-raf-1−/− (−/−, closed bars) littermates. A representative experiment out of two is shown. Download figure Download PowerPoint Increased apoptosis in c-raf-1−/− embryonic fibroblasts The data reported above show that c-raf-1−/− fetal liver cells cultured in vitro fail to accumulate and are more prone than wild-type cells to undergo apoptosis. We next established primary fibroblasts from c-raf-1−/− fetuses. The morphology and size of the mutant fibroblasts were indistinguishable from those of wild-type cells. How ever, the numbers of mutant cells obtained from these cultures were significantly lower compared with heterozygous or wild-type controls (Figure 5A). In addition, c-raf-1−/− fibroblasts showed lower saturation densities than wild-type cells at all serum concentrations, ranging from 5 to 30% (∼70% of controls). The saturation densities of mutant and wild-type fibroblasts increased proportionally with serum concentration. Thus, the mutant cells are still able to respond to growth factors, albeit to a reduced extent (Figure 5B). Raf-1−/− fibroblasts could be immortalized, although they underwent prolonged crisis (data not shown). If cell proliferation was affected, the lack of Raf-1 should delay G1 to S progression during the cell cycle. However, fluorescence-activated cell sorting (FACS) analysis of asynchronous cells revealed that G1, S and G2 cell cycle phases were distributed similarly in c-raf-1−/− and wild-type fibroblasts (Figure 5C and D). To confirm this observation, we synchronized primary fibroblasts in G0 by density arrest and growth factor withdrawal, and measured the percentage of cells with a DNA content >2N after serum stimulation. Both c-raf-1−/− and wild-type fibroblasts exited G1 with the same kinetics. As a control, wild-type fibroblasts failed to progress through the cell cycle in the presence of a MEK inhibitor (Figure 5E). Figure 5.Characterization of c-raf-1−/− fibroblast cultures. (A) Proliferation curves of wild-type (+/+), c-raf-1+/− (+/−) and c-raf-1−/− (−/−) primary fibroblasts. The average ± SD numbers of cells isolated from two individual littermates of each genotype are shown, each determined in triplicate. (B) Saturation density analysis of wild-type (+/+, open bars) and c-raf-1−/− (−/−, closed bars) primary fibroblasts as a function of serum concentration. Cells were counted after 14 days of culture in the presence of the indicated concentrations of FCS. The average ± SD numbers of cells isolated from two individual littermates of each genotype are shown, each determined in triplicate. (C and D) Representative FACS profiles of continuosly growing control (+/+) and c-raf-1−/− (−/−) primary fibroblasts. Asynchronous cells were stained with propidium iodide and their DNA content was determined by FACS analysis. (E) Quantitative analysis of G1/S-phase progression in wild-type (+/+) primary fibroblasts, untreated or treated with the MEK inhibitor PD 98059 (50 μM, throughout the duration of the experiment) and c-raf-1−/− (−/−) primary fibroblasts. The percentage of cells exiting G1 (i.e. the percentage of cells in S, G2 or M) at the indicated times after serum stimulation of G0-synchronized cells (see Materials and methods) was quantified by propidium iodide staining and FACS analysis. (F) Percentage of BrdU-incorporating and TUNEL-positive cells in parallel asynchronous cultures of primary wild-type (+/+, open bars) and Raf-1−/− (−/−, closed bars) fibroblasts. The data represent the mean value of cells isolated from two individual littermates of each genotype, and vertical bars represent the range of the samples. (G) Effect of different apoptosis-inducing stimuli on fibroblasts from wild-type (open bars) and c-raf-1−/− (closed bars) littermates. Primary fibroblasts (left panel) were serum starved or treated with 20 ng/ml actinomycin D (ActD) for 24 h. 3T3-like cells (right panel) were treated with 20 ng/ml actinomycin D and 50 ng/ml hamster anti-mouse Fas antibody for 22 h, or with 100 ng/ml murine TNF-α, 5 μg/ml cycloheximide (CHX) or a combination of both for 12 h. Cell death was assessed by measuring lactate dehydrogenase release. Primary fibroblasts or 3T3-like cells isolated from three individual littermates of each genotype were used in the experiment, and the data shown represent the mean ± SD. Download figure Download PowerPoint The FACS profiles of continuously growing cultures showed an increased number of cells with a sub-2N DNA content in Raf-1−/− fibroblasts as compared with wild type (Figure 5C and D; 5.26 versus 2.43%), suggesting increased apoptosis in the G1 phase. The consequences of Raf-1 inactivation for fibroblast turnover were assessed directly by determining simultaneously the number of cells in S phase [by bromodeoxyuridine (BrdU) labeling] and the number of apoptotic cells (by TUNEL staining). Consistent with the results summarized above, the number of S-phase cells in wild-type and mutant cultures was indistinguishable. The number of apoptotic cells in the Raf-1−/− cultures, however, was clearly elevated as compared with wild type (Figure 5F; 8.65 ± 3.20% in mutant versus 1.83 ± 0.02% in wild-type cultures). Thus, the reduced cell yield of c-raf-1−/− cultures correlates with an increase in apoptosis, but not with a cell cycle defect. In addition, primary Raf-1−/− fibroblasts were more susceptible than wild type to apoptosis induced by growth factor deprivation and actinomycin D treatment (Figure 5G, left panel). Raf-1−/− fibroblasts immortalized according to the 3T3 protocol lost their hypersensitivity towards actinomycin D (Figure 5G, right panel), but were more susceptible than wild-type cells to Fas activation. In contrast, 3T3-like Raf-1-deficient cells were resistant to the cytotoxic effects of tumor necrosis factor-α (TNF-α) alone, and they were not more susceptible than the wild type to a combined treatment with TNF-α and cycloheximide. Therefore, Raf-1 inactivation appears to cause hypersensitivity to selective apoptotic stimuli. Activation of the ERK pathway and I-κB degradation are unaffected in c-raf-1−/− fibroblasts We next investigated whether the inactivation of c-raf-1 had any effect on the ERK pathway. Epidermal growth factor (EGF)-stimulated MEK kinase activity was still present in whole-cell lysates of Raf-1−/− fibroblasts, but it was strongly reduced (∼30% of wild type; Figure 6A). These data indicate that Raf-1 represents a major fraction of the cellular MEK kinase activity in vitro, and that its loss is not compensated by the up-regulation of other MEK kinases. B-Raf is expressed at a much lower level than Raf-1 in fibroblasts, and compensatory overexpression was not observed in Raf-1−/− cells (Figure 6B). However, the basal as well as the EGF-stimulated activity of B-Raf were elevated (2-fold) in Raf-1-deficient fibroblasts compared with wild-type cells (Figure 6C). Activation of MEK and MAPK was normal in Raf-1−/− fibroblasts treated with a variety of extracellular stimuli (Figure 6D). The responses to EGF (Figure 6E), analyzed in more detail, showed the same intensity and kinetics of stimulation in wild-type and mutant cells. Furthermore, lysates of whole c-raf-1−/−, c-raf-1+/− and c-raf-1+/+ littermate embryos contained indistinguishable amounts of phosphorylated ERK (Figure 6F). Thus, Raf-1 is dispensable for the activation of the ERK pathway in fibroblasts and in the whole embryo. Figure 6.Activation of the ERK pathway and of the B-Raf kinase in c-raf-1−/− fibroblasts. (A) The MEK kinase activity present in whole-cell lysates of untreated or EGF-stimulated (33 nM, 5 min) wild-type (+/+) or c-raf-1−/− (−/−) primary fibroblasts was measured in a coupled assay. (B) Raf immunoblot of whole-cell lysates from wild-type (+/+), c-raf-1+/− (+/−) or c-raf-1−/− (−/−) primary fibroblasts. The antibody used was raised against the Raf kinase domain and recognizes all three Raf proteins, albeit with different efficiency (Raf-1 >> B-Raf >> A-Raf; Kolch et al., 1990). An actin immunoblot is shown as a loading control. (C) Kinase activity of B-Raf immunoprecipitates measured in a coupled assay. The amounts of B-Raf contained in the immunoprecipitates were equal, as determined by immunoblotting (not shown). The results are expressed as c.p.m. incorporated into the substrate. One representative experiment out of three is shown. (D and E) Wild-type (+/+) and c-raf-1−/− (−/−) primary fibroblasts were stimulated for 10 min with EGF (33 nM), FCS (10%) or TPA (5 μM) (D), or with 33 nM EGF for different time periods (E) prior to cell lysis. The presence of the phosphorylated, active forms of MEK (pMEK) and ERK (pERK), as well as of MEK2 as a loading control, was detected by immunoblotting with the corresponding antibodies. (F) ERK phosphorylation in lysates from wild-type (+/+), c-raf-1+/− (+/−) or c-raf-1−/− (−/−) embryos. A Raf-1 immunoblot is shown as a genotype control and a MEK-2 immunoblot is shown as a loading control. Download figure Download PowerPoint We next monitored ERK activation in fibroblasts treated with apoptotic stimuli. ERK phosphorylation occurred normally in Raf-1-deficient cells treated with Fas antibody or with TNF-α (Figure 7). Figure 7.ERK phosphorylation and I-κB degradation in c-raf-1−/− fibroblasts treated with anti-Fas antibody or TNF-α. Wild-type (+/+) and c-raf-1−/− (−/−) primary fibroblasts were stimulated with 50 ng/ml hamster anti-mouse Fas antibody (A) or 100 ng/ml TNF-α (B) for different time periods prior to cell lysis. The presence of the phos phorylated, active forms of ERK (pERK), as well as of I-κB or of the loading control MEK2, was detected by immunoblotting with the corresponding antibodies. Download figure Download PowerPoint A further downstream target of Raf-1 implicated in protection from apoptosis is the transcription factor NF-κB (Foo and Nolan, 1999). Raf-1 activates NF-κB by inducing I-κB phosphorylation and degradation. This pathway is distinct from MEK/ERK activation, but involves MEKK-1 upstream of the I-κB kinase complex (Baumann et al., 2000). Treatment of fibrobla