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Protective role of Raf-1 in Salmonella-induced macrophage apoptosis.

Jesenberger V, Procyk KJ, Rüth J, Schreiber M, Theussl HC, Wagner EF, Baccarini M - J. Exp. Med. (2001)

Bottom Line: Macrophages lacking c-raf-1 are hypersensitive towards pathogen-induced apoptosis.Surprisingly, activation of the antiapoptotic mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and nuclear factor kappaB pathways is normal in Raf-1-deficient macrophages, and mitochondrial fragility is not increased.Instead, pathogen-mediated activation of caspase-1 is enhanced selectively, implying that Raf-1 antagonizes stimulus-induced caspase-1 activation and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Microbiology, Institute of Microbiology and Genetics, Vienna Biocenter, Austria.

ABSTRACT
Invasive Salmonella induces macrophage apoptosis via the activation of caspase-1 by the bacterial protein SipB. Here we show that infection of macrophages with Salmonella causes the activation and degradation of Raf-1, an important intermediate in macrophage proliferation and activation. Raf-1 degradation is SipB- and caspase-1-dependent, and is prevented by proteasome inhibitors. To study the functional significance of Raf-1 in this process, the c-raf-1 gene was inactivated by Cre-loxP-mediated recombination in vivo. Macrophages lacking c-raf-1 are hypersensitive towards pathogen-induced apoptosis. Surprisingly, activation of the antiapoptotic mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and nuclear factor kappaB pathways is normal in Raf-1-deficient macrophages, and mitochondrial fragility is not increased. Instead, pathogen-mediated activation of caspase-1 is enhanced selectively, implying that Raf-1 antagonizes stimulus-induced caspase-1 activation and apoptosis.

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Related in: MedlinePlus

Conditional targeting of the mouse c-raf-1 gene. (A) Schematic representation of the conditional targeting of the c-raf-1 gene. Genomic, genomic locus before recombination; floxNeoTK, homologously recombined targeting vector; flox, targeted locus after Cre-mediated removal of the NeoTK cassette; and c-raf-1Δ, c-raf-1 locus deleted in bone marrow cells after in vivo induction of Mx-Cre. loxP sites (▸) were inserted 5′ and 3′ of exon 3 of the c-raf-1 gene. Selection markers (a neomycin resistance gene for positive selection and the thymidine kinase gene of herpes virus for negative selection) were positioned between two loxP sites upstream of the floxed exon 3. The PstI sites delimiting the fragments obtained by digesting the genomic and mutated c-raf-1 alleles are marked. The PstI site upstream of the 3′ loxP site was introduced with the targeting vector and serves as a marker for the floxed allele. (B) Southern blot analysis of PstI-digested genomic DNA from targeted ES cell clones after transient Cre expression. The probe used is shown in black in A. The Neo/TK gene cassette was excised by transiently expressing Cre. Excision was confirmed by Southern blot analysis with a Neo/TK probe (data not shown). (C) Southern blot analysis of PstI-digested genomic DNA isolated from bone marrow cells of c-raf-1flox/flox;MxCre− and c-raf-1Δ/Δ;MxCre+ littermates after induction of Mx-Cre in vivo. Genomic DNA from a c-raf-1flox/− animal was used to mark the position of the c-raf-1 alleles. (D) Western blot analysis of whole cell lysates from bone marrow–derived macrophages. Macrophages derived from c-raf-1flox/flox;MxCre− mice and from c-raf-1flox/flox;Cre+ littermates treated in vivo with poly I:C were infected with invasive Salmonella (moi 25) for 20 min. The amount of Raf-1 in whole cell lysates was determined by immunoblotting.
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Figure 3: Conditional targeting of the mouse c-raf-1 gene. (A) Schematic representation of the conditional targeting of the c-raf-1 gene. Genomic, genomic locus before recombination; floxNeoTK, homologously recombined targeting vector; flox, targeted locus after Cre-mediated removal of the NeoTK cassette; and c-raf-1Δ, c-raf-1 locus deleted in bone marrow cells after in vivo induction of Mx-Cre. loxP sites (▸) were inserted 5′ and 3′ of exon 3 of the c-raf-1 gene. Selection markers (a neomycin resistance gene for positive selection and the thymidine kinase gene of herpes virus for negative selection) were positioned between two loxP sites upstream of the floxed exon 3. The PstI sites delimiting the fragments obtained by digesting the genomic and mutated c-raf-1 alleles are marked. The PstI site upstream of the 3′ loxP site was introduced with the targeting vector and serves as a marker for the floxed allele. (B) Southern blot analysis of PstI-digested genomic DNA from targeted ES cell clones after transient Cre expression. The probe used is shown in black in A. The Neo/TK gene cassette was excised by transiently expressing Cre. Excision was confirmed by Southern blot analysis with a Neo/TK probe (data not shown). (C) Southern blot analysis of PstI-digested genomic DNA isolated from bone marrow cells of c-raf-1flox/flox;MxCre− and c-raf-1Δ/Δ;MxCre+ littermates after induction of Mx-Cre in vivo. Genomic DNA from a c-raf-1flox/− animal was used to mark the position of the c-raf-1 alleles. (D) Western blot analysis of whole cell lysates from bone marrow–derived macrophages. Macrophages derived from c-raf-1flox/flox;MxCre− mice and from c-raf-1flox/flox;Cre+ littermates treated in vivo with poly I:C were infected with invasive Salmonella (moi 25) for 20 min. The amount of Raf-1 in whole cell lysates was determined by immunoblotting.

Mentions: Disruption of the c-raf-1 gene is embryonic lethal at midgestation and is accompanied by fetal liver apoptosis (unpublished observations). To obtain Raf-1–deficient macrophages, conditional inactivation of the c-raf-1 gene was achieved by the insertion of loxP sites cloned 5′ and 3′ of exon 3. A selection cassette (a neomycin resistance gene for positive selection and the thymidine kinase gene of herpes virus for negative selection) was positioned between two loxP sites upstream of the floxed exon 3 (Fig. 3 A). The mutation was introduced into ES cells by homologous recombination. After transient Cre expression, ES cell clones in which the Neo/TK gene cassette, but not exon 3, had been excised were identified by Southern blot analysis (Fig. 3 B). Germline-transmitting chimeras were obtained and bred to C57Bl/6 mice. Mice homozygous for the c-raf-1flox allele were phenotypically indistinguishable from wt animals. To allow inducible inactivation of Raf-1, c-raf-1flox/flox animals were crossed to mice expressing the Cre recombinase under the control of the Mx1 promoter 31. Injection of mice with poly I:C resulted in the efficient deletion of the floxed exon 3 in liver (data not shown) and bone marrow (Fig. 3 C, c-raf-1Δ/Δ). Macrophages derived from these bone marrow cells were devoid of Raf-1 protein, as shown by Western blot analysis, while poly I:C treatment of c-raf-1flox/flox animals that did not carry the Mx-Cre transgene did not have any effect on Raf-1 expression (Fig. 3 D).


Protective role of Raf-1 in Salmonella-induced macrophage apoptosis.

Jesenberger V, Procyk KJ, Rüth J, Schreiber M, Theussl HC, Wagner EF, Baccarini M - J. Exp. Med. (2001)

Conditional targeting of the mouse c-raf-1 gene. (A) Schematic representation of the conditional targeting of the c-raf-1 gene. Genomic, genomic locus before recombination; floxNeoTK, homologously recombined targeting vector; flox, targeted locus after Cre-mediated removal of the NeoTK cassette; and c-raf-1Δ, c-raf-1 locus deleted in bone marrow cells after in vivo induction of Mx-Cre. loxP sites (▸) were inserted 5′ and 3′ of exon 3 of the c-raf-1 gene. Selection markers (a neomycin resistance gene for positive selection and the thymidine kinase gene of herpes virus for negative selection) were positioned between two loxP sites upstream of the floxed exon 3. The PstI sites delimiting the fragments obtained by digesting the genomic and mutated c-raf-1 alleles are marked. The PstI site upstream of the 3′ loxP site was introduced with the targeting vector and serves as a marker for the floxed allele. (B) Southern blot analysis of PstI-digested genomic DNA from targeted ES cell clones after transient Cre expression. The probe used is shown in black in A. The Neo/TK gene cassette was excised by transiently expressing Cre. Excision was confirmed by Southern blot analysis with a Neo/TK probe (data not shown). (C) Southern blot analysis of PstI-digested genomic DNA isolated from bone marrow cells of c-raf-1flox/flox;MxCre− and c-raf-1Δ/Δ;MxCre+ littermates after induction of Mx-Cre in vivo. Genomic DNA from a c-raf-1flox/− animal was used to mark the position of the c-raf-1 alleles. (D) Western blot analysis of whole cell lysates from bone marrow–derived macrophages. Macrophages derived from c-raf-1flox/flox;MxCre− mice and from c-raf-1flox/flox;Cre+ littermates treated in vivo with poly I:C were infected with invasive Salmonella (moi 25) for 20 min. The amount of Raf-1 in whole cell lysates was determined by immunoblotting.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195927&req=5

Figure 3: Conditional targeting of the mouse c-raf-1 gene. (A) Schematic representation of the conditional targeting of the c-raf-1 gene. Genomic, genomic locus before recombination; floxNeoTK, homologously recombined targeting vector; flox, targeted locus after Cre-mediated removal of the NeoTK cassette; and c-raf-1Δ, c-raf-1 locus deleted in bone marrow cells after in vivo induction of Mx-Cre. loxP sites (▸) were inserted 5′ and 3′ of exon 3 of the c-raf-1 gene. Selection markers (a neomycin resistance gene for positive selection and the thymidine kinase gene of herpes virus for negative selection) were positioned between two loxP sites upstream of the floxed exon 3. The PstI sites delimiting the fragments obtained by digesting the genomic and mutated c-raf-1 alleles are marked. The PstI site upstream of the 3′ loxP site was introduced with the targeting vector and serves as a marker for the floxed allele. (B) Southern blot analysis of PstI-digested genomic DNA from targeted ES cell clones after transient Cre expression. The probe used is shown in black in A. The Neo/TK gene cassette was excised by transiently expressing Cre. Excision was confirmed by Southern blot analysis with a Neo/TK probe (data not shown). (C) Southern blot analysis of PstI-digested genomic DNA isolated from bone marrow cells of c-raf-1flox/flox;MxCre− and c-raf-1Δ/Δ;MxCre+ littermates after induction of Mx-Cre in vivo. Genomic DNA from a c-raf-1flox/− animal was used to mark the position of the c-raf-1 alleles. (D) Western blot analysis of whole cell lysates from bone marrow–derived macrophages. Macrophages derived from c-raf-1flox/flox;MxCre− mice and from c-raf-1flox/flox;Cre+ littermates treated in vivo with poly I:C were infected with invasive Salmonella (moi 25) for 20 min. The amount of Raf-1 in whole cell lysates was determined by immunoblotting.
Mentions: Disruption of the c-raf-1 gene is embryonic lethal at midgestation and is accompanied by fetal liver apoptosis (unpublished observations). To obtain Raf-1–deficient macrophages, conditional inactivation of the c-raf-1 gene was achieved by the insertion of loxP sites cloned 5′ and 3′ of exon 3. A selection cassette (a neomycin resistance gene for positive selection and the thymidine kinase gene of herpes virus for negative selection) was positioned between two loxP sites upstream of the floxed exon 3 (Fig. 3 A). The mutation was introduced into ES cells by homologous recombination. After transient Cre expression, ES cell clones in which the Neo/TK gene cassette, but not exon 3, had been excised were identified by Southern blot analysis (Fig. 3 B). Germline-transmitting chimeras were obtained and bred to C57Bl/6 mice. Mice homozygous for the c-raf-1flox allele were phenotypically indistinguishable from wt animals. To allow inducible inactivation of Raf-1, c-raf-1flox/flox animals were crossed to mice expressing the Cre recombinase under the control of the Mx1 promoter 31. Injection of mice with poly I:C resulted in the efficient deletion of the floxed exon 3 in liver (data not shown) and bone marrow (Fig. 3 C, c-raf-1Δ/Δ). Macrophages derived from these bone marrow cells were devoid of Raf-1 protein, as shown by Western blot analysis, while poly I:C treatment of c-raf-1flox/flox animals that did not carry the Mx-Cre transgene did not have any effect on Raf-1 expression (Fig. 3 D).

Bottom Line: Macrophages lacking c-raf-1 are hypersensitive towards pathogen-induced apoptosis.Surprisingly, activation of the antiapoptotic mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and nuclear factor kappaB pathways is normal in Raf-1-deficient macrophages, and mitochondrial fragility is not increased.Instead, pathogen-mediated activation of caspase-1 is enhanced selectively, implying that Raf-1 antagonizes stimulus-induced caspase-1 activation and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Microbiology, Institute of Microbiology and Genetics, Vienna Biocenter, Austria.

ABSTRACT
Invasive Salmonella induces macrophage apoptosis via the activation of caspase-1 by the bacterial protein SipB. Here we show that infection of macrophages with Salmonella causes the activation and degradation of Raf-1, an important intermediate in macrophage proliferation and activation. Raf-1 degradation is SipB- and caspase-1-dependent, and is prevented by proteasome inhibitors. To study the functional significance of Raf-1 in this process, the c-raf-1 gene was inactivated by Cre-loxP-mediated recombination in vivo. Macrophages lacking c-raf-1 are hypersensitive towards pathogen-induced apoptosis. Surprisingly, activation of the antiapoptotic mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and nuclear factor kappaB pathways is normal in Raf-1-deficient macrophages, and mitochondrial fragility is not increased. Instead, pathogen-mediated activation of caspase-1 is enhanced selectively, implying that Raf-1 antagonizes stimulus-induced caspase-1 activation and apoptosis.

Show MeSH
Related in: MedlinePlus