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Protein kinase Cepsilon is required for macrophage activation and defense against bacterial infection.

Castrillo A, Pennington DJ, Otto F, Parker PJ, Owen MJ, Boscá L - J. Exp. Med. (2001)

Bottom Line: Macrophages from PKCepsilon(-/-) animals demonstrated a severely attenuated response to lipopolysaccharide (LPS) and interferon (IFN)gamma, characterized by a dramatic reduction in the generation of NO, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta.This study provides direct evidence that PKCepsilon is critically involved at an early stage of LPS-mediated signaling in activated macrophages.Furthermore, we demonstrate that in the absence of PKCepsilon, host defense against bacterial infection is severely compromised, resulting in an increased incidence of mortality.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Cientificas-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain.

ABSTRACT
To assess directly the role of protein kinase C (PKC)epsilon in the immune system, we generated mice that carried a homozygous disruption of the PKCepsilon locus. PKCepsilon(-/-) animals appeared normal and were generally healthy, although female mice frequently developed a bacterial infection of the uterus. Macrophages from PKCepsilon(-/-) animals demonstrated a severely attenuated response to lipopolysaccharide (LPS) and interferon (IFN)gamma, characterized by a dramatic reduction in the generation of NO, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta. Further analysis revealed that LPS-stimulated macrophages from PKCepsilon(-/-) mice were deficient in the induction of nitric oxide synthase (NOS)-2, demonstrating a decrease in the activation of IkappaB kinase, a reduction in IkappaB degradation, and a decrease in nuclear factor (NF)kappaB nuclear translocation. After intravenous administration of Gram-negative or Gram-positive bacteria, PKCepsilon(-/-) mice demonstrated a significantly decreased period of survival. This study provides direct evidence that PKCepsilon is critically involved at an early stage of LPS-mediated signaling in activated macrophages. Furthermore, we demonstrate that in the absence of PKCepsilon, host defense against bacterial infection is severely compromised, resulting in an increased incidence of mortality.

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The activation of NF-κB and IKK2 are attenuated in LPS-stimulated macrophages from PKCε−/− mice. (A) Thioglycollate-elicited peritoneal macrophages were activated with 200 ng/ml of LPS, and at the indicated times cytosolic and nuclear protein extracts were prepared. NF-κB activity was determined by EMSA after the binding of nuclear proteins to the distal κB sequence of the murine NOS-2 promoter. The nature of the retained bands was determined by supershift assays (not shown). Normalization was accomplished using the binding of proteins to the PPARα site of acyl CoA oxidase. (B) IκBα levels were determined by Western blot, and the amount of phosphorylated (S32)IκBα was evaluated in cells treated with MG 132 (20 μM), and using a specific anti-phospho(S32)-IκBα Ab. (C) Macrophages were elicited and treated as above, and at the indicated time points IKK2 was immunoprecipitated from the cytosolic extracts. IKK2 activity was assayed using GST-IκBα (amino acids 1–54) and [γ-32P]ATP as substrates. Results show the incorporation of [32P]phosphate into the substrate, with an aliquot of IKK2 assayed by Western blot to evaluate the level of enzyme.
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fig5: The activation of NF-κB and IKK2 are attenuated in LPS-stimulated macrophages from PKCε−/− mice. (A) Thioglycollate-elicited peritoneal macrophages were activated with 200 ng/ml of LPS, and at the indicated times cytosolic and nuclear protein extracts were prepared. NF-κB activity was determined by EMSA after the binding of nuclear proteins to the distal κB sequence of the murine NOS-2 promoter. The nature of the retained bands was determined by supershift assays (not shown). Normalization was accomplished using the binding of proteins to the PPARα site of acyl CoA oxidase. (B) IκBα levels were determined by Western blot, and the amount of phosphorylated (S32)IκBα was evaluated in cells treated with MG 132 (20 μM), and using a specific anti-phospho(S32)-IκBα Ab. (C) Macrophages were elicited and treated as above, and at the indicated time points IKK2 was immunoprecipitated from the cytosolic extracts. IKK2 activity was assayed using GST-IκBα (amino acids 1–54) and [γ-32P]ATP as substrates. Results show the incorporation of [32P]phosphate into the substrate, with an aliquot of IKK2 assayed by Western blot to evaluate the level of enzyme.

Mentions: Much evidence exists to suggest that the upregulation of NOS-2 requires the transcription factor NFκB (23, 25). Macrophages are known to express NFκB constitutively, but in resting cells NFκB forms an inactive cytoplasmic complex with IκB. On activation, IκB is phosphorylated and degraded, leading to the release of NFκB, which translocates to the nucleus as an active transcription factor. To assess whether the reduction of NOS-2 expression in PKCε−/− mice was due to a decrease in NFκB activation, the presence of active NFκB in macrophage nuclear extracts was analyzed by electrophoretic mobility shift assay (EMSA) after increasing length of LPS stimulation of thioglycollate-elicited cells. The distal κB sequences from the NOS-2 promoter were used to bind the nuclear extracts. In comparison to the clear binding of NFκB (p50.p65) in wild-type lysates, NFκB levels in nuclear extracts from PKCε−/− mice were greatly reduced at all time points analyzed (Fig. 5 A). Supershift assays were performed to verify the nature of the observed bands (data not shown). These results suggest that the failure to activate NFκB in PKCε−/− macrophages could be responsible for the low levels of NOS-2 expression observed in response to LPS stimulation.


Protein kinase Cepsilon is required for macrophage activation and defense against bacterial infection.

Castrillo A, Pennington DJ, Otto F, Parker PJ, Owen MJ, Boscá L - J. Exp. Med. (2001)

The activation of NF-κB and IKK2 are attenuated in LPS-stimulated macrophages from PKCε−/− mice. (A) Thioglycollate-elicited peritoneal macrophages were activated with 200 ng/ml of LPS, and at the indicated times cytosolic and nuclear protein extracts were prepared. NF-κB activity was determined by EMSA after the binding of nuclear proteins to the distal κB sequence of the murine NOS-2 promoter. The nature of the retained bands was determined by supershift assays (not shown). Normalization was accomplished using the binding of proteins to the PPARα site of acyl CoA oxidase. (B) IκBα levels were determined by Western blot, and the amount of phosphorylated (S32)IκBα was evaluated in cells treated with MG 132 (20 μM), and using a specific anti-phospho(S32)-IκBα Ab. (C) Macrophages were elicited and treated as above, and at the indicated time points IKK2 was immunoprecipitated from the cytosolic extracts. IKK2 activity was assayed using GST-IκBα (amino acids 1–54) and [γ-32P]ATP as substrates. Results show the incorporation of [32P]phosphate into the substrate, with an aliquot of IKK2 assayed by Western blot to evaluate the level of enzyme.
© Copyright Policy
Related In: Results  -  Collection

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fig5: The activation of NF-κB and IKK2 are attenuated in LPS-stimulated macrophages from PKCε−/− mice. (A) Thioglycollate-elicited peritoneal macrophages were activated with 200 ng/ml of LPS, and at the indicated times cytosolic and nuclear protein extracts were prepared. NF-κB activity was determined by EMSA after the binding of nuclear proteins to the distal κB sequence of the murine NOS-2 promoter. The nature of the retained bands was determined by supershift assays (not shown). Normalization was accomplished using the binding of proteins to the PPARα site of acyl CoA oxidase. (B) IκBα levels were determined by Western blot, and the amount of phosphorylated (S32)IκBα was evaluated in cells treated with MG 132 (20 μM), and using a specific anti-phospho(S32)-IκBα Ab. (C) Macrophages were elicited and treated as above, and at the indicated time points IKK2 was immunoprecipitated from the cytosolic extracts. IKK2 activity was assayed using GST-IκBα (amino acids 1–54) and [γ-32P]ATP as substrates. Results show the incorporation of [32P]phosphate into the substrate, with an aliquot of IKK2 assayed by Western blot to evaluate the level of enzyme.
Mentions: Much evidence exists to suggest that the upregulation of NOS-2 requires the transcription factor NFκB (23, 25). Macrophages are known to express NFκB constitutively, but in resting cells NFκB forms an inactive cytoplasmic complex with IκB. On activation, IκB is phosphorylated and degraded, leading to the release of NFκB, which translocates to the nucleus as an active transcription factor. To assess whether the reduction of NOS-2 expression in PKCε−/− mice was due to a decrease in NFκB activation, the presence of active NFκB in macrophage nuclear extracts was analyzed by electrophoretic mobility shift assay (EMSA) after increasing length of LPS stimulation of thioglycollate-elicited cells. The distal κB sequences from the NOS-2 promoter were used to bind the nuclear extracts. In comparison to the clear binding of NFκB (p50.p65) in wild-type lysates, NFκB levels in nuclear extracts from PKCε−/− mice were greatly reduced at all time points analyzed (Fig. 5 A). Supershift assays were performed to verify the nature of the observed bands (data not shown). These results suggest that the failure to activate NFκB in PKCε−/− macrophages could be responsible for the low levels of NOS-2 expression observed in response to LPS stimulation.

Bottom Line: Macrophages from PKCepsilon(-/-) animals demonstrated a severely attenuated response to lipopolysaccharide (LPS) and interferon (IFN)gamma, characterized by a dramatic reduction in the generation of NO, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta.This study provides direct evidence that PKCepsilon is critically involved at an early stage of LPS-mediated signaling in activated macrophages.Furthermore, we demonstrate that in the absence of PKCepsilon, host defense against bacterial infection is severely compromised, resulting in an increased incidence of mortality.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Cientificas-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain.

ABSTRACT
To assess directly the role of protein kinase C (PKC)epsilon in the immune system, we generated mice that carried a homozygous disruption of the PKCepsilon locus. PKCepsilon(-/-) animals appeared normal and were generally healthy, although female mice frequently developed a bacterial infection of the uterus. Macrophages from PKCepsilon(-/-) animals demonstrated a severely attenuated response to lipopolysaccharide (LPS) and interferon (IFN)gamma, characterized by a dramatic reduction in the generation of NO, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta. Further analysis revealed that LPS-stimulated macrophages from PKCepsilon(-/-) mice were deficient in the induction of nitric oxide synthase (NOS)-2, demonstrating a decrease in the activation of IkappaB kinase, a reduction in IkappaB degradation, and a decrease in nuclear factor (NF)kappaB nuclear translocation. After intravenous administration of Gram-negative or Gram-positive bacteria, PKCepsilon(-/-) mice demonstrated a significantly decreased period of survival. This study provides direct evidence that PKCepsilon is critically involved at an early stage of LPS-mediated signaling in activated macrophages. Furthermore, we demonstrate that in the absence of PKCepsilon, host defense against bacterial infection is severely compromised, resulting in an increased incidence of mortality.

Show MeSH
Related in: MedlinePlus