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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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Raf-1 degradation is a consequence of macrophage apoptosis and is inhibited by proteasome inhibitors. (A) Bone marrow–derived macrophages were infected with either wt or sipB− Salmonella. (B) Primary macrophages were isolated from control (+/+) or caspase-1–deficient mice (caspase-1−/−) and infected with invasive Salmonella. The amount of Raf-1 in Triton X-100 extracts of whole cells was determined by immunoblotting. (C) Macrophages were infected with invasive bacteria. The cells were lysed at the indicated times after infection, and the Triton X-100 (Tx-100) soluble fractions (30 μg, 1/10 of total) and the whole insoluble fractions were analyzed by immunoblotting with a Raf-1 antiserum. (D) Macrophages were treated with a caspase inhibitor (200 μM ZVAD-fmk) or with proteasome inhibitors (10 μM MG-132 and 10 μM MG-115) for 90 min before infection with Salmonella for 15 min. The amount of Raf-1 in the detergent-soluble and -insoluble fractions of whole cell lysates was determined by immunoblotting.
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Figure 2: Raf-1 degradation is a consequence of macrophage apoptosis and is inhibited by proteasome inhibitors. (A) Bone marrow–derived macrophages were infected with either wt or sipB− Salmonella. (B) Primary macrophages were isolated from control (+/+) or caspase-1–deficient mice (caspase-1−/−) and infected with invasive Salmonella. The amount of Raf-1 in Triton X-100 extracts of whole cells was determined by immunoblotting. (C) Macrophages were infected with invasive bacteria. The cells were lysed at the indicated times after infection, and the Triton X-100 (Tx-100) soluble fractions (30 μg, 1/10 of total) and the whole insoluble fractions were analyzed by immunoblotting with a Raf-1 antiserum. (D) Macrophages were treated with a caspase inhibitor (200 μM ZVAD-fmk) or with proteasome inhibitors (10 μM MG-132 and 10 μM MG-115) for 90 min before infection with Salmonella for 15 min. The amount of Raf-1 in the detergent-soluble and -insoluble fractions of whole cell lysates was determined by immunoblotting.

Mentions: Next we investigated if the decrease in the amount of Raf-1 observed during the late phases of infection was dependent on caspase-1, and whether it was a reflection of the general demise of the cell undergoing apoptosis or rather a specific phenomenon involving this kinase selectively. Triton X-100 extracts from control (+/+) or caspase-1–deficient macrophages infected with Salmonella were immunoblotted with a Raf-1 antiserum. The amount of kinase was progressively reduced in +/+ macrophages starting from 5 min after infection with invasive Salmonella, but remained constant throughout infection with a sipB− mutant (Fig. 2 A) or of caspase-1–deficient macrophages with wt bacteria (Fig. 2 B). In contrast to Raf-1, the amount of MEK (data not shown) or ERK remained constant throughout infection. Thus, Salmonella induced caspase-1–dependent degradation of Raf-1, but not of MEK or ERK (Fig. 2A and Fig. B).


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)

Raf-1 degradation is a consequence of macrophage apoptosis and is inhibited by proteasome inhibitors. (A) Bone marrow–derived macrophages were infected with either wt or sipB− Salmonella. (B) Primary macrophages were isolated from control (+/+) or caspase-1–deficient mice (caspase-1−/−) and infected with invasive Salmonella. The amount of Raf-1 in Triton X-100 extracts of whole cells was determined by immunoblotting. (C) Macrophages were infected with invasive bacteria. The cells were lysed at the indicated times after infection, and the Triton X-100 (Tx-100) soluble fractions (30 μg, 1/10 of total) and the whole insoluble fractions were analyzed by immunoblotting with a Raf-1 antiserum. (D) Macrophages were treated with a caspase inhibitor (200 μM ZVAD-fmk) or with proteasome inhibitors (10 μM MG-132 and 10 μM MG-115) for 90 min before infection with Salmonella for 15 min. The amount of Raf-1 in the detergent-soluble and -insoluble fractions of whole cell lysates was determined by immunoblotting.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195927&req=5

Figure 2: Raf-1 degradation is a consequence of macrophage apoptosis and is inhibited by proteasome inhibitors. (A) Bone marrow–derived macrophages were infected with either wt or sipB− Salmonella. (B) Primary macrophages were isolated from control (+/+) or caspase-1–deficient mice (caspase-1−/−) and infected with invasive Salmonella. The amount of Raf-1 in Triton X-100 extracts of whole cells was determined by immunoblotting. (C) Macrophages were infected with invasive bacteria. The cells were lysed at the indicated times after infection, and the Triton X-100 (Tx-100) soluble fractions (30 μg, 1/10 of total) and the whole insoluble fractions were analyzed by immunoblotting with a Raf-1 antiserum. (D) Macrophages were treated with a caspase inhibitor (200 μM ZVAD-fmk) or with proteasome inhibitors (10 μM MG-132 and 10 μM MG-115) for 90 min before infection with Salmonella for 15 min. The amount of Raf-1 in the detergent-soluble and -insoluble fractions of whole cell lysates was determined by immunoblotting.
Mentions: Next we investigated if the decrease in the amount of Raf-1 observed during the late phases of infection was dependent on caspase-1, and whether it was a reflection of the general demise of the cell undergoing apoptosis or rather a specific phenomenon involving this kinase selectively. Triton X-100 extracts from control (+/+) or caspase-1–deficient macrophages infected with Salmonella were immunoblotted with a Raf-1 antiserum. The amount of kinase was progressively reduced in +/+ macrophages starting from 5 min after infection with invasive Salmonella, but remained constant throughout infection with a sipB− mutant (Fig. 2 A) or of caspase-1–deficient macrophages with wt bacteria (Fig. 2 B). In contrast to Raf-1, the amount of MEK (data not shown) or ERK remained constant throughout infection. Thus, Salmonella induced caspase-1–dependent degradation of Raf-1, but not of MEK or ERK (Fig. 2A and Fig. B).

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