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Lipopolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn.

Meng F, Lowell CA - J. Exp. Med. (1997)

Bottom Line: Nitrite production and cytokine secretion (IL-1, IL-6, and TNF-alpha) are normal or even enhanced in hck-/-fgr-/-lyn-/- macrophages after LPS stimulation.Furthermore, the activation of the ERK1/2 and JNK kinases, as well as the transcription factor NF-kappaB, are the same in normal and mutant macrophages after LPS stimulation.The current study provides direct evidence that three Src-family kinases Hck, Fgr, and Lyn are not obligatory for LPS-initiated signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, University of California, San Francisco 94143-0724, USA.

ABSTRACT
Lipopolysaccharide (LPS) stimulates immune responses by interacting with the membrane receptor CD14 to induce the generation of cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6. The mechanism by which the LPS signal is transduced from the extracellular environment to the nuclear compartment is not well defined. Recently, an increasing amount of evidence suggests that protein tyrosine kinases especially the Src-family kinases Hck, Fgr, and Lyn, play important roles in LPS signaling. To directly address the physiological function of Hck, Fgr and Lyn in LPS signaling, a genetic approach has been used to generate mutations of all three kinases in a single mouse strain. hck-/-fgr-/-lyn-/- mice are moderately healthy and fertile; macrophages cultured from these mice express normal levels of CD14 and no other Src-family kinases were detected. Although the total protein phosphotyrosine level is greatly reduced in macrophages derived from hck-/-fgr-/-lyn-/- mice, functional analyses indicate that both elicited peritoneal (PEMs) and bone marrow-derived macrophages (BMDMs) from triple mutant mice have no major defects in LPS-induced activation. Nitrite production and cytokine secretion (IL-1, IL-6, and TNF-alpha) are normal or even enhanced in hck-/-fgr-/-lyn-/- macrophages after LPS stimulation. The development of tumor cell cytotoxicity is normal in triple mutant BMDMs and only partially impaired in PEMs after LPS stimulation. Furthermore, the activation of the ERK1/2 and JNK kinases, as well as the transcription factor NF-kappaB, are the same in normal and mutant macrophages after LPS stimulation. The current study provides direct evidence that three Src-family kinases Hck, Fgr, and Lyn are not obligatory for LPS-initiated signal transduction.

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Activation of NF-κB binding activity by LPS/IFN-γ. PEM  and BMDM (5 × 106 cells/assay) from wild-type and hck−/−fgr−/−lyn−/−  mice were cultured with or without LPS (10 ng/ml) and IFN-γ (20 ng/ml)  for 2 h. Nuclear extracts were prepared and incubated with a α-[32P]dCTP– labeled double-stranded oligonucleotide, corresponding to the murine  immunoglobulin κ enhancer site. Protein-bound oligonucleotides were  resolved by electrophoresis. Specific binding by NF-κB was confirmed by  using an oligonucleotide containing mutations in the NF-κB binding  motif. S, specific binding to wild-type oligonucleotide; NS, non-specific  binding to oligonucleotide containing point mutations.
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Figure 8: Activation of NF-κB binding activity by LPS/IFN-γ. PEM and BMDM (5 × 106 cells/assay) from wild-type and hck−/−fgr−/−lyn−/− mice were cultured with or without LPS (10 ng/ml) and IFN-γ (20 ng/ml) for 2 h. Nuclear extracts were prepared and incubated with a α-[32P]dCTP– labeled double-stranded oligonucleotide, corresponding to the murine immunoglobulin κ enhancer site. Protein-bound oligonucleotides were resolved by electrophoresis. Specific binding by NF-κB was confirmed by using an oligonucleotide containing mutations in the NF-κB binding motif. S, specific binding to wild-type oligonucleotide; NS, non-specific binding to oligonucleotide containing point mutations.

Mentions: Another major signaling pathway that is initiated in macrophages after LPS treatment is activation of NF-κB DNA binding activity by dissociation from Iκ-B (37). To assess whether the loss of Hck, Fgr, and Lyn affected NF-κB activation after LPS stimulation, we performed electrophoretic mobility shift assays (EMSA) using nuclear extracts from untreated and LPS-treated cells incubated with a double-stranded oligonucleotide containing an NF-κB recognition sequence (38). After LPS/IFN-γ stimulation for 2 h, we detected inducible NF-κB DNA binding activity in PEMs and BMDMs derived from both wild-type and hck−/−fgr−/−lyn−/− macrophages (Fig. 8). No binding was observed using an oligonucleotide containing point mutations in the NF-κB recognition sequence. The results demonstrate that the deficiency of Hck, Fgr, and Lyn did not effect NF-κB activation by LPS.


Lipopolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn.

Meng F, Lowell CA - J. Exp. Med. (1997)

Activation of NF-κB binding activity by LPS/IFN-γ. PEM  and BMDM (5 × 106 cells/assay) from wild-type and hck−/−fgr−/−lyn−/−  mice were cultured with or without LPS (10 ng/ml) and IFN-γ (20 ng/ml)  for 2 h. Nuclear extracts were prepared and incubated with a α-[32P]dCTP– labeled double-stranded oligonucleotide, corresponding to the murine  immunoglobulin κ enhancer site. Protein-bound oligonucleotides were  resolved by electrophoresis. Specific binding by NF-κB was confirmed by  using an oligonucleotide containing mutations in the NF-κB binding  motif. S, specific binding to wild-type oligonucleotide; NS, non-specific  binding to oligonucleotide containing point mutations.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196288&req=5

Figure 8: Activation of NF-κB binding activity by LPS/IFN-γ. PEM and BMDM (5 × 106 cells/assay) from wild-type and hck−/−fgr−/−lyn−/− mice were cultured with or without LPS (10 ng/ml) and IFN-γ (20 ng/ml) for 2 h. Nuclear extracts were prepared and incubated with a α-[32P]dCTP– labeled double-stranded oligonucleotide, corresponding to the murine immunoglobulin κ enhancer site. Protein-bound oligonucleotides were resolved by electrophoresis. Specific binding by NF-κB was confirmed by using an oligonucleotide containing mutations in the NF-κB binding motif. S, specific binding to wild-type oligonucleotide; NS, non-specific binding to oligonucleotide containing point mutations.
Mentions: Another major signaling pathway that is initiated in macrophages after LPS treatment is activation of NF-κB DNA binding activity by dissociation from Iκ-B (37). To assess whether the loss of Hck, Fgr, and Lyn affected NF-κB activation after LPS stimulation, we performed electrophoretic mobility shift assays (EMSA) using nuclear extracts from untreated and LPS-treated cells incubated with a double-stranded oligonucleotide containing an NF-κB recognition sequence (38). After LPS/IFN-γ stimulation for 2 h, we detected inducible NF-κB DNA binding activity in PEMs and BMDMs derived from both wild-type and hck−/−fgr−/−lyn−/− macrophages (Fig. 8). No binding was observed using an oligonucleotide containing point mutations in the NF-κB recognition sequence. The results demonstrate that the deficiency of Hck, Fgr, and Lyn did not effect NF-κB activation by LPS.

Bottom Line: Nitrite production and cytokine secretion (IL-1, IL-6, and TNF-alpha) are normal or even enhanced in hck-/-fgr-/-lyn-/- macrophages after LPS stimulation.Furthermore, the activation of the ERK1/2 and JNK kinases, as well as the transcription factor NF-kappaB, are the same in normal and mutant macrophages after LPS stimulation.The current study provides direct evidence that three Src-family kinases Hck, Fgr, and Lyn are not obligatory for LPS-initiated signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, University of California, San Francisco 94143-0724, USA.

ABSTRACT
Lipopolysaccharide (LPS) stimulates immune responses by interacting with the membrane receptor CD14 to induce the generation of cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6. The mechanism by which the LPS signal is transduced from the extracellular environment to the nuclear compartment is not well defined. Recently, an increasing amount of evidence suggests that protein tyrosine kinases especially the Src-family kinases Hck, Fgr, and Lyn, play important roles in LPS signaling. To directly address the physiological function of Hck, Fgr and Lyn in LPS signaling, a genetic approach has been used to generate mutations of all three kinases in a single mouse strain. hck-/-fgr-/-lyn-/- mice are moderately healthy and fertile; macrophages cultured from these mice express normal levels of CD14 and no other Src-family kinases were detected. Although the total protein phosphotyrosine level is greatly reduced in macrophages derived from hck-/-fgr-/-lyn-/- mice, functional analyses indicate that both elicited peritoneal (PEMs) and bone marrow-derived macrophages (BMDMs) from triple mutant mice have no major defects in LPS-induced activation. Nitrite production and cytokine secretion (IL-1, IL-6, and TNF-alpha) are normal or even enhanced in hck-/-fgr-/-lyn-/- macrophages after LPS stimulation. The development of tumor cell cytotoxicity is normal in triple mutant BMDMs and only partially impaired in PEMs after LPS stimulation. Furthermore, the activation of the ERK1/2 and JNK kinases, as well as the transcription factor NF-kappaB, are the same in normal and mutant macrophages after LPS stimulation. The current study provides direct evidence that three Src-family kinases Hck, Fgr, and Lyn are not obligatory for LPS-initiated signal transduction.

Show MeSH
Related in: MedlinePlus