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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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Related in: MedlinePlus

The generation and characterization of PKCε−/− mice. (A) Schematic representation of the PKCε locus knockout strategy, illustrating the insertion of a lacZ/Neo cassette into the PstI site in the first exon of the PKCε gene. The predicted EcoRI fragments and probe for Southern blot analysis of the wild-type and mutant alleles are shown. (B) Southern blot analysis of homozygous mutant, heterozygous and wild-type tail DNA from a representative litter. (C) RT-PCR on brain RNA from two wild-type and two PKCε−/− mice. RT-PCR for β-actin is shown as loading control. (D) Western blot analysis for PKC family members on brain tissue from PKCε−/− and control mice.
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fig1: The generation and characterization of PKCε−/− mice. (A) Schematic representation of the PKCε locus knockout strategy, illustrating the insertion of a lacZ/Neo cassette into the PstI site in the first exon of the PKCε gene. The predicted EcoRI fragments and probe for Southern blot analysis of the wild-type and mutant alleles are shown. (B) Southern blot analysis of homozygous mutant, heterozygous and wild-type tail DNA from a representative litter. (C) RT-PCR on brain RNA from two wild-type and two PKCε−/− mice. RT-PCR for β-actin is shown as loading control. (D) Western blot analysis for PKC family members on brain tissue from PKCε−/− and control mice.

Mentions: A 9.3-kb EcoRI fragment containing the first exon of the PKCε gene was isolated from a murine 129/Sv genomic DNA library (gift from T. Rabbitts, Laboratory of Molecular Biology, Cambridge). A targeting vector was generated by introducing a positive selectable cassette into the PstI site of exon 1. This cassette contains stop codons in all three frames, an independent ribosomal entry site (IRES) followed by the LacZ gene with an SV40 polyadenylation sequence, and a neomycin phosphotransferase gene (MC1Neo poly(A); gift from A. Smith, Centre for Genome Research, Edinburgh, UK). Correctly targeted GK129 ES cells (see Fig. 1 for Southern blot details) were injected into C57BL/6 blastocysts. Chimeric mice were bred to C57BL/6 mice in specific pathogen-free (SPF) conditions to generate PKCε homozygous mutant animals. PKCε−/− and PKCε+/− mice were maintained on a mixed C57Bl/6 × 129/Sv genetic background and were genotyped by Southern blot (Fig. 1, A and B) or PCR analysis of tail DNA (data not shown). Mice were fed ad libitum with a standard diet (Panlab) and kept under a light and dark cycle of 12 h (lights on at 8 a.m.).


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 generation and characterization of PKCε−/− mice. (A) Schematic representation of the PKCε locus knockout strategy, illustrating the insertion of a lacZ/Neo cassette into the PstI site in the first exon of the PKCε gene. The predicted EcoRI fragments and probe for Southern blot analysis of the wild-type and mutant alleles are shown. (B) Southern blot analysis of homozygous mutant, heterozygous and wild-type tail DNA from a representative litter. (C) RT-PCR on brain RNA from two wild-type and two PKCε−/− mice. RT-PCR for β-actin is shown as loading control. (D) Western blot analysis for PKC family members on brain tissue from PKCε−/− and control mice.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195971&req=5

fig1: The generation and characterization of PKCε−/− mice. (A) Schematic representation of the PKCε locus knockout strategy, illustrating the insertion of a lacZ/Neo cassette into the PstI site in the first exon of the PKCε gene. The predicted EcoRI fragments and probe for Southern blot analysis of the wild-type and mutant alleles are shown. (B) Southern blot analysis of homozygous mutant, heterozygous and wild-type tail DNA from a representative litter. (C) RT-PCR on brain RNA from two wild-type and two PKCε−/− mice. RT-PCR for β-actin is shown as loading control. (D) Western blot analysis for PKC family members on brain tissue from PKCε−/− and control mice.
Mentions: A 9.3-kb EcoRI fragment containing the first exon of the PKCε gene was isolated from a murine 129/Sv genomic DNA library (gift from T. Rabbitts, Laboratory of Molecular Biology, Cambridge). A targeting vector was generated by introducing a positive selectable cassette into the PstI site of exon 1. This cassette contains stop codons in all three frames, an independent ribosomal entry site (IRES) followed by the LacZ gene with an SV40 polyadenylation sequence, and a neomycin phosphotransferase gene (MC1Neo poly(A); gift from A. Smith, Centre for Genome Research, Edinburgh, UK). Correctly targeted GK129 ES cells (see Fig. 1 for Southern blot details) were injected into C57BL/6 blastocysts. Chimeric mice were bred to C57BL/6 mice in specific pathogen-free (SPF) conditions to generate PKCε homozygous mutant animals. PKCε−/− and PKCε+/− mice were maintained on a mixed C57Bl/6 × 129/Sv genetic background and were genotyped by Southern blot (Fig. 1, A and B) or PCR analysis of tail DNA (data not shown). Mice were fed ad libitum with a standard diet (Panlab) and kept under a light and dark cycle of 12 h (lights on at 8 a.m.).

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