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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: 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.

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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Analysis of ERK1/2  and JNK activation after LPS/ IFN-γ stimulation. PEMs and  BMDMs from wild-type and  hck−/−fgr−/−lyn−/− mutant mice  were stimulated with LPS (10  ng/ml) and IFN-γ (20 ng/ml) for  the indicated time periods, then  total cell lysates prepared. ERK1/2  and JNK proteins were immunoprecipitated (250 μg of cell lysates)  using ERK1/2 and JNK polyclonal antibodies, respectively,  and tested for kinase activity in  the presence of γ-[33P]ATP using MBP and C-Jun fusion protein, respectively, as substrates.  Phosphorylated proteins were  resolved on 12% SDS-PAGE.
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Figure 7: Analysis of ERK1/2 and JNK activation after LPS/ IFN-γ stimulation. PEMs and BMDMs from wild-type and hck−/−fgr−/−lyn−/− mutant mice were stimulated with LPS (10 ng/ml) and IFN-γ (20 ng/ml) for the indicated time periods, then total cell lysates prepared. ERK1/2 and JNK proteins were immunoprecipitated (250 μg of cell lysates) using ERK1/2 and JNK polyclonal antibodies, respectively, and tested for kinase activity in the presence of γ-[33P]ATP using MBP and C-Jun fusion protein, respectively, as substrates. Phosphorylated proteins were resolved on 12% SDS-PAGE.

Mentions: To evaluate the role of Hck, Fgr, and Lyn in LPS-induced activation of MAPKs, we determined ERK1/2 and JNK kinase activity using defined substrates. ERK1/2 kinase activity was determined by phosphorylation of MBP. In both PEMs and BMDMs, ERK1/2 kinase activity was activated after LPS/IFN-γ treatment in hck−/−fgr−/−lyn−/− cells compared to wild-type cells (Fig. 7). No consistent difference in ERK1/2 activation was observed between wild-type and mutant cells. The activation of ERK1/2 was detected at 60 min and declined at 2 h after LPS and IFN-γ stimulation.


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)

Analysis of ERK1/2  and JNK activation after LPS/ IFN-γ stimulation. PEMs and  BMDMs from wild-type and  hck−/−fgr−/−lyn−/− mutant mice  were stimulated with LPS (10  ng/ml) and IFN-γ (20 ng/ml) for  the indicated time periods, then  total cell lysates prepared. ERK1/2  and JNK proteins were immunoprecipitated (250 μg of cell lysates)  using ERK1/2 and JNK polyclonal antibodies, respectively,  and tested for kinase activity in  the presence of γ-[33P]ATP using MBP and C-Jun fusion protein, respectively, as substrates.  Phosphorylated proteins were  resolved on 12% SDS-PAGE.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196288&req=5

Figure 7: Analysis of ERK1/2 and JNK activation after LPS/ IFN-γ stimulation. PEMs and BMDMs from wild-type and hck−/−fgr−/−lyn−/− mutant mice were stimulated with LPS (10 ng/ml) and IFN-γ (20 ng/ml) for the indicated time periods, then total cell lysates prepared. ERK1/2 and JNK proteins were immunoprecipitated (250 μg of cell lysates) using ERK1/2 and JNK polyclonal antibodies, respectively, and tested for kinase activity in the presence of γ-[33P]ATP using MBP and C-Jun fusion protein, respectively, as substrates. Phosphorylated proteins were resolved on 12% SDS-PAGE.
Mentions: To evaluate the role of Hck, Fgr, and Lyn in LPS-induced activation of MAPKs, we determined ERK1/2 and JNK kinase activity using defined substrates. ERK1/2 kinase activity was determined by phosphorylation of MBP. In both PEMs and BMDMs, ERK1/2 kinase activity was activated after LPS/IFN-γ treatment in hck−/−fgr−/−lyn−/− cells compared to wild-type cells (Fig. 7). No consistent difference in ERK1/2 activation was observed between wild-type and mutant cells. The activation of ERK1/2 was detected at 60 min and declined at 2 h after LPS and IFN-γ stimulation.

Bottom Line: 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.

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